Immunization through oral administration of a vaccine with an edible product

ABSTRACT

A vaccine produced in edible plant and/or animal products, as well as a method of second-generation vaccine development through the production of at least one complete structure of a pathogen in a transgenic plant or animal is described. Preferably, the present invention enables the production of virus-like particles in edible food plants, through the co-expression of a plurality of proteins and/or of a plurality of portions of such proteins. The co-expression of viral structural proteins should enhance the immunogenicity of the transgenic antigen by providing all elements necessary to induce a protective immune response. In one variation of the method the immune response to the transgenic vaccine would be induced by presenting the transgenic proteins as part of an edible plant structure, utilizing this to stimulate the mucosal immune system. Alternatively, the vaccine produced in transgenic plant/animal products could be purified and used as an immunogen in other vaccination strategies. A corollary to this claim is the use of transgenic plant-derived viral vaccines as recombinant vectors to shuttle host or therapeutic genes to the liver or other tissues of the body utilizing the natural trophism of the virus. A second method of vaccination, that involves the stimulation of mucosal immunity, includes the use of conventional vaccines that are applied to the surface of the rectum to induce an immune response that is manifest both locally (in the intestinal mucosa) and systemically (blood and other body tissues).

[0001] This Application is a Continuation-in-Part Application of PCT Application No. PCT/IL01/00550, filed on Jun. 15, 2001; and of U.S. patent application Ser. No. 09/596,060, filed on Jun. 16, 2000 (now U.S. Pat. No. 6,368,602); both of which are hereby incorporated by reference as if fully set forth herein.

FIELD OF THE INVENTION

[0002] The present invention relates to a method of immunizing a subject against a pathogen through administration of a vaccine with an edible product, and in particular, to such a method in which the vaccine is created through a genetically modified plant as a component of the edible tissue of the plant. Preferably, the vaccine is directed against Hepatitis A virus (HAV). The present invention also relates to the use of conventional HAV vaccines for mucosal immunization by application of the vaccine to the rectal or nasal mucosal surface, optionally in the form of a suppository or other such formulation, for the purpose of inducing an immune response to the virus that protects against hepatitis A. The present invention also relates to the immunotherapeutic use of virus like particles that are composed of viral structural proteins forming a capsid or a virus like structure.

BACKGROUND OF THE INVENTION

[0003] Infectious diseases are a worldwide cause of morbidity and mortality. Those particularly at risk are individuals with weaker immune systems such as very young children or those with congenital and acquired immunodeficiencies. Despite the impact of vaccination programs for many of the pathogens, infectious diseases are still the primary cause of childhood mortality, worldwide (The World Health Report 1997). Travel and other contact between populations have increased the risk for spread of bacterial and viral pathogens, thereby demanding a higher degree of community protection than ever before. The most efficient and effective strategy for preventing these diseases is by mass vaccination (Review; Vaccine supplement, Nature Medicine 1998;4:474-534). However, despite significant technological progress in bioengineering and advancements in large-scale production, vaccines remain costly and often, unavailable in sufficient quantity and/or supply to conduct mass vaccination programs. Currently, most vaccines are administered parenterally by injection. Further, such vaccines must be kept refrigerated until administered. Thus, mass vaccination programs currently require a large supply of vaccine, maintenance of the cold chain, and supplies of sterile syringes and needles. These limitations make population immunization campaigns difficult to execute in some jurisdictions, particularly in Third World countries, or in emergency response situations such as threatened use of a pathogen in biological warfare or bioterrorism.

[0004] HAV (Genus: Hepatovirus, Family: picornaviriade) is an example of a pathogen for which the vaccine is currently difficult to administer through such mass vaccination programs. As it is a highly communicable and infectious, water or food-borne pathogen, HAV has also been recognized by the US Centers for Disease Control (CDC) as a pathogen with bioterrorism potential (WHO/CDS/CSR/EDC/2000.7, Hepatitis A, World Health Organization, Dept. of Communicable Disease Surveillance and Response document. http//www.who.int/emc). HAV is highly contagious and transmitted principally by the fecal-oral route (Koff R. S. Hepatitis A. Lancet 1998; 351:164349) but it may also be acquired by sexual (anal-oral) contact and blood transfusions. The course of hepatitis A is highly variable and age-dependent. In the very young, cases of hepatitis A are usually asymptomatic and can only be detected by identifying biochemical or serologic changes in the blood. However, asymptomatic individuals still shed large amounts of virus in their feces, and therefore, are a significant reservoir for disease spread. The severity of disease increases with age of the patient and symptoms range from mild/transient—to severe/prolonged—to fulminant fatal hepatitis. The clinical course of symptomatic hepatitis A typically displays: an asymptomatic preclinical incubation period of 10-50 days when viral shedding is maximal; a prodromal (preicteric) phase lasting approx. a week characterized by loss of appetite, fatigue, fever, gastrointestinal symptoms, dark urine and pale stools; and an icteric phase with jaundice (bilirubin>20-40 mg/L). This latter phase usually coincides with the onset of the immune response (at approx. 10 days after infection) and the reduction of viremia. Nevertheless, viral shedding into the stools may continue for several weeks. Occasionally (more often in patients who are more than 50 years old), fulminant hepatitis with severe liver necrosis, high fever, hepatic encephalopathy with coma and seizures may occur, leading to death in 70-90% of such cases. Co-infection with hepatitis C or B complicates outcome and reduces prognosis of recovery. However, in the majority of cases the disease is self-limiting, convalescence long, and recovery occurs with bed rest. Often convalescent patients experience fatigue for up to one year. Relapsing hepatitis occurs in 3-20% of cases (usually, within 4-15 weeks) and persistent cholesteric hepatitis with high bilirubin levels is occasionally observed. There are no specific treatments for hepatitis A and current methods of prevention are described below.

[0005] There is only one known HAV serotype and a single immunodominant conformation antigenic site (described above) to which virus-neutralizing antibodies are directed (WHO/CDS/CSR/EDC/2000.7, Hepatitis A. World Health Organization, Department of Communicable Disease Surveillance and Response document. http:/www.who.int/emc; Gauss-Muller V, Zhou M Q, von der Helm K, Deinhardt F. Recombinant proteins VP1 and VP3 of hepatitis A prime for neutralizing antibody response. J Med Virol 1990;31;277-83; Wang C D, Tschen S -Y, Heinricy U, et al. Immune response to hepatitis A virus capsids after infection. J Clin Microbiol 1996; 34:707-13). While antibodies to individual structural and nonstructural HAV proteins may be detected, they have no known protective function. Therefore, intact whole viral particles are always used for immunization. In acute HAV infection both IgM and IgG class antibodies are detected and are diagnostic. HAV-specific IgM may persist for up to 6 months. Appearance of specific IgG is associated with reduction of viremia and acquisition of lifelong immunity to HAV. Liver disease symptoms, which appear at the onset of the immune response, have been attributed to cellular immunity rather than to immune complex-mediated tissue damage or to direct viral cytopathogenic effects (Viral Infections in Humans, Epidemiology and Control (3^(rd) Edition). Evans A S, editor. New York, Plenum Medical Book Co., 1998; Karayiannis P, Jowett T, Enticott M, et al. Hepatitis A virus replication in tamarins and host immune response in relation to pathogenesis of liver damage. J Med Virol 1986; 18:261-76, Kurane I, Binn L N, Bancroft W H, Ennis F A Human lymphocyte responses to hepatitis A virus-infected cells: interferon production and lysis of infected cells. J Immunol 1985; 135:2140-4).

[0006] Currently, there are at least four HAV vaccines that have received regulatory approval and are in current use (Prevention of hepatitis A through active or passive immunization. Recommendations of the Advisory Committee on Immunization Practices (ACIP). CDC, MMWR 1999, 48(RR1-12):1-37.). All consist of whole formalin-inactivated HAV that has been grown in various primate cell lines and purified. As viral titers are inevitably low these vaccines are in limited supply, expensive and therefore are subject to exclusive use for controlling disease outbreaks or for immunizing travelers to hepatitis A-endemic areas. Hence, present HAV vaccines can only be purchased for small-scale programs and not for mass immunization. Further, as such vaccines may be administered only by injection, syringes, needles, and trained staff must be available, adding to the cost and difficulty of delivery and limiting population uptake for those who have fear of needles. Clearly, new solutions for cheap and effective mass vaccination are needed. Providing a vaccine through a simple method could significantly increase vaccine uptake and population protection against HAV. Two possible solutions are: the delivery of HAV vaccine as an edible product (i.e., a transgenic plant-produced HAV vaccine); or in the form of a suppository or similar formulation that may be applied to the surface of the nasal cavity or rectum. These methods would induce immunity to HAV via stimulation of the mucosal immune system.

[0007] The mucosal immune system, an interconnected network of lymphocytes and accessory cells located throughout mucosae of the oral and nasal cavities, bronchi, gut, urinary and genital systems, functions both nonspecifically and specifically as a first line of defense against pathogens entering the body via these routes. It has both inductive sites (located in lymphoid follicles of nasal and rectal mucosae, and Peyer's patches located on the serosal surface of the small intestine) and effector molecules (which include secretory IgA produced by lymphocytes located throughout the mucosae, and cytokines elaborated by CD4+ helper T-cells) as well as cytotoxic CD8+ T-cells and intraepithelial leukocytes (IEL) which are cellular effectors of mucosal immunity. While it is separate from the systemic immune system (spleen, thymus, lymph nodes and peripheral blood lymphocytes), it is interconnected as its immune cells also circulate systemically. Vaccination studies have shown that mucosal immunization provokes both local and systemic immunity, while injection of antigen usually leads only to systemic immunity. Moreover, as the mucosal immune system is interconnected, vaccination at one mucosal site (such as the nasal cavity) can lead to expression of immunity at a distant mucosal site (such as the rectum or vagina), as well as systemically. Hence, mucosal immunization has a greater potential for inducing effective immunity. Mucosal immunization is also relatively easy and inexpensive to undertake as a means of mass vaccination as it can be executed without special training or equipment. Since currently used HAV vaccines are administered systemically (by injection) and not locally (to the gut mucosa) they may not be particularly effective at inducing immunity where it is initially needed—i.e., at the gastrointestinal mucosa where the virus first penetrates and where it possibly replicates in the early stages of disease. In contrast, vaccines that are applied locally to the gut are likely to be more effective at inducing T-cell and antibody responses both locally and systemically (Mestecky J, Fultz P N, Mucosal immune system of the human genital tract. J Infect Dis 1999; 179:S470-S474; Lehner T, Wang Y, Ping L, et al. The effect of route of immunization on mucosal immunity and protection. J Infect Dis 1999; 179:S489-S492).

[0008] Recently, it was demonstrated that vaccination against bacteria, such as salmonella, or viral pathogens, such as HIV, may possibly be enhanced through the rectal administration of attenuated or killed bacteria/viruses (see, for example: Nardelli-Haefliger D, Kraehenbuhl J P, Curtiss R 3rd, et al. Oral and rectal immunization of adult female volunteers with a recombinant attenuated Salmonella typhi vaccine strain, Infect Immun. 1996;64:5219-24; Lehner T. Bergmeier L, Wang Y, Tao L, Mitchell E. A rational basis for mucosal vaccination against HIV infection. Immunol Rev. 1999 ; 170;183-96). Rectal, nasal or oral administrations of vaccines have been shown to elicit both humoral and cellular immune responses that protect against infection. Results have varied according to the type of antigen, adjuvant (materials which enhance the immune response), dose, and route used. A highly successful oral immunogen is the Sabin polio vaccine that has been shown to induce high levels of protective virus-neutralizing antibodies.

[0009] Many human pathogens enter the body via mucosal routes such as the surfaces of the gastrointestinal, oral-nasal, genitourinary tracts. Hence, mucosal immunization, which has the potential to stimulate both local (mucosal) and systemic immunity, is theoretically, the preferred strategy. Unfortunately, at the present time HAV and many other vaccines are delivered only by injection, limiting their uptake and utility. Many of these might be adaptable to oral, rectal and nasal immunization strategies. Even for those vaccines that are available in an orally administered form, however, mass vaccination programs are difficult to perform because of storage and handling requirements for the vaccines, as well as the cost and availability. A thermostable, inexpensive-to-produce product which is readily produced in large quantities would be the solution to the world's current and emergency response vaccine needs. In an attempt to overcome some of these problems, several groups have produced genetically engineered plants capable of expressing bacterial or viral proteins. For example, cholera toxin B subunit oligomers were expressed in transgenic potato plants, although the plants were not tested for immunogenicity (Arakawa et al., Expression of Cholera Toxin B Subunit Oligomers in Transgenic Potato Plants. Transgenic Research, 6:403413; 1997). Similarly, the rabies virus glycoprotein, which coats the outer surface of the virus, has also been expressed in transgenic tomato plants, although again, the plants were not tested for immunogenicity (McGarvey et al. Expression of the Rabies Virus Glycoprotein in Transgenic Tomatoes. Biotechnology, 13:1484-1487, 1995). Also, U.S. Pat. No. 5,914,123 describes vaccines that are expressed in plants, but also does not provide any data concerning the immunogenicity of these vaccines. U.S. Pat. Nos. 5,612,487 and 5,484,719 also describe plant-derived anti-viral vaccines but similarly, do not provide immunogenicity data. Not all plant-derived vaccines that have been evaluated in vaccination studies have proven to be immunogenic. In many cases normal oral consumption of the plant product only triggered a partially protective immune response in animals and humans (Mason et al. Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice. PNAS, 93:5335-5340, 1996).

[0010] A partial explanation for the low immunogenicity of many of these plant-based vaccines is that the targeted antigens have been expressed as isolated proteins or peptides, and not in the context of the intact pathogen. Hence these transgenic antigens cannot assume the correct conformation for induction of neutralizing antibodies. For example, HAV capsid proteins expressed individually do not elicit neutralize antibodies. This is not surprising, as earlier studies showed that other viruses and bacteria exhibit similar behavior when only portions of the overall structure are administered as conventional vaccines (Almond and Heeney. AIDS vaccine development in primate models. AIDS 12(suppl A):S133-140, 1998; Mayr et al. Development of replication-defective adenovirus serotope 5 containing the capsid and 3C protease coding regions of Foot-and-Mouth Disease virus as a vaccine candidate. Virology, 263:496506, 1999; and Wigdorovitz et al. Induction of a protective antibody response to Foot-and-Mouth Disease virus in mice following oral or parenteral immunization with alfalfa transgenic plants expressing the viral structural protein VP1. Virology, 255;347-353, 1999). However, in other cases oral immunization has proven successful in animal models and in human volunteers (Xiang Z, Ertl H C J. Induction of mucosal immunity with a replication-defective adenoviral recombinant. Vaccine 1999; 17:2003-8 [a report comparing nasal, rectal, vaginal and injection methods of immunization with a rabies subunit vaccine produced as an adenovirus recombinant]; Kapusta J, Moedelska A, Figlerowicz, et al. A plant-derived edible vaccine against hepatitis B virus. FASEB J 1999; 13:1796-99 [a report concerning a HAV subunit vaccine produced in transgenic plants and administered by feeding]. These observations indicate that virus-like particles or some subunit antigens administered orally are capable of inducing an immune response that may be protective; yet these same reports indicate that different systems of antigens generate apparently different levels of protection and immune responsiveness. However, these reports do not concern intact viral particles produced in transgenic plants.

[0011] Oral administration of viral antigens may also be used to induce specific tolerance, which may be a desired outcome of immunization. Previous studies in which viral subunits (hepatitis B surface antigen, HbsAg) were used in oral immunization showed that this strategy reduced inflammation caused by viral infection (see for example; Ilan Y, Chowdhury J R. Induction of tolerance to hepatitis B virus: can we “eat the disease” and live with the virus? Med Hypotheses 1999;52(6):505-9 [a report concerning the induction of tolerance to EBsAg to transform patients with severe chronic active hepatitis B into healthy HAV carriers]; Ilan Y, Sauter B, Chowdhury N R, et al. Oral tolerization to adenoviral proteins permits repeated adenovirus-mediated gene therapy in rats with pre-existing to adenoviruses. Hepatology 1998;27 (5):1368-76 [a study concerning oral administration of replication defective recombinant adenovirus generated in mammalian tissue culture for the purpose of inducing oral tolerance to abrogate the host immune response to adenovirus and to prolong adenovirus mediated gene therapy]. The results of these studies support, that tolerance to viral antigens may be induced through oral administration of viral subunits, or whole virus. However, none of the studies concern virus or viral subunits that have been generated in a transgenic plant system, nor do they demonstrate the induction of tolerance to multiple viral antigens presented in a chimeric format. Also, none of the reports describe the induction of tolerance with engineered viral particles.

SUMMARY OF THE INVENTION

[0012] The background art does not teach or suggest a vaccine that is produced by edible plant and/or animal materials, for regular consumption (that is, through oral administration), which contains at least one complete structure of a disease-causing pathogen. The background art also does not teach or suggest that such a complete structure may optionally be a plurality of proteins, or alternatively may comprise a single molecule that mimics the structure of pathogen. The background art also does not teach or suggest a successfully immunogenic vaccine for viruses such as HAV. Also, the background art does not teach or suggest a method for producing such vaccines in transgenic plants and/or animals. The background art does not teach that a chimeric virus like particle containing elements of two pathogens may be used to induce specific immunologic tolerance that may be exploited for immunotherapeutic purposes—i.e., to reduce tissue inflammation induced by a pathogenic protein, or to downregulate the immune response to allow the persistence of a viral vector used to transfer new genes into host tissue.

[0013] There is thus, a need for, and it would be useful to have, a vaccine that is able to successfully elicit a protective immune response to disease-causing pathogens, through the production of at least one complete structure of the pathogen, regardless of whether the pathogen is viral, bacterial or parasitic in nature.

[0014] There is also a need for an immunotherapeutic vaccine to serve as a method of enhancing immunologic unresponsiveness to viral proteins that may be involved in inducing host tissue inflammation, or as a method of enhancing the persistence of viral vectored genes in host tissue in gene therapy.

[0015] The present invention overcomes these problems of the background art, and also provides a solution to a long-felt need for producing vaccines in edible plant and/or animal products, by providing a new developmental method for second generation protective or immunotherapeutic vaccines or gene vectors, through the production of at least one complete structure of a pathogen in a transgenic plant or animal, as well as by providing the vaccines themselves. Preferably, the present invention enables the production of virus-like particles in edible food plants, through the co-expression of a plurality of proteins and/or of a plurality of portions of such proteins as recombinant peptide structures capable of attaining sufficient immunogenic conformational structure to give rise to an immune response that will protect against, and/or ameliorate disease.

[0016] As a preferred example of the operation of the present invention, a vaccine was developed for hepatitis A virus (HAV). Previous attempts to vaccinate with isolated HAV capsid proteins failed to elicit a protective immune response, because isolated HAV proteins are incapable of eliciting neutralizing antibodies that recognize specific conformational structures on the viral particle created only after the assembly of the capsid. To overcome this problem, preferably the present invention includes the construction of two HAV plasmids carrying a non-infectious HAV genome lacking the 5′UTR, for stable transformation of plants. In one plasmid (pGPPatΩHAV) transcription of HAV is driven from the patatin promoter for expression in tomato fruits, and in the second plasmid (pJDHAV) transcription is under the control of the 35SCaMV promoter and should be expressed in the green parts of transgenic plants. To induce an immune response to the HAV transgenes, the edible parts of transgenic plants are ingested. Assessment of the immunogenic potential of these vaccines is by feeding of transgenic plant material to experimental animals.

[0017] According to preferred embodiments of the present invention, the technology that is developed for vaccines can also optionally and more preferably be applied for immunotherapy to block or dampen immune-mediated inflammatory tissue responses or for gene targeting to specific organs through the consumption of edible plant components containing known infectious viral particles as shuttles for the genes of interest. As described below, the present invention is optionally able to induce tolerance, which may preferably be used to reduce an immune response selectively, for example for gene therapy.

[0018] Using HAV as a model pathogen, the present invention also embodies a method for administering a regular HAV vaccine to a subject by absorption through a mucosal tissue, particularly through the mucosa of the rectum, but also including the nasal mucosa. This method enables the HAV vaccine to be administered to the subject rectally or nasally, for example as a suppository or other dosage form, to successfully immunize the subject against HAV. Thus, the methods of present invention overcome problems of background art methods of administration that are associated with current methods of vaccination against HAV—viz, availability and supply of inactivated whole viral vaccines, expense, maintenance of the vaccine in proper storage conditions that typically include refrigeration, need for special equipment and expertise for vaccine delivery. As edible or topically applied mucosal vaccines may be self-administered, large-scale immunization programs would be feasible.

[0019] With regard to a vaccine administered through mucosal tissue, as described in greater detail below, rectal immunization of mice with HAVRIX™, an alum adjuvanted whole inactivated HAV vaccine induced strong HAV-specific IgA and cellular proliferative responses both systemically and in the intestinal mucosa that were significantly greater than those elicited by intraperitoneal delivery of the same vaccine. A further potential advantage of mucosal immunization is that response to natural reimmunization through oral ingestion of pathogens may be stronger than that after parenteral vaccination.

[0020] Hereinafter, the digestive system is defined as mouth, esophagus, stomach, small intestine, large intestine and rectum, or any portion thereof.

[0021] Hereinafter, the term “treatment” of a disease and/or condition also optionally includes prevention of the disease and/or condition thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The foregoing and other objects, aspects and advantages will be better understood from the following detailed description of a preferred embodiment of the invention with reference to the drawings, wherein;

[0023]FIG. 1A is a schematic diagram for describing the generation of plant-derived VLP (virus-like particles). As shown, the structural core proteins are preferably expressed in plant material, more preferably through stable transfection and expression of the relevant gene(s) in the plant material. These proteins then preferably assemble to form a capsid-like structure, or virus-like particle, which may either optionally be empty (right side of figure) or may alternatively contain a gene (left side of figure), for example for performing gene therapy.

[0024]FIG. 1B is a schematic diagram of the HAV-transfection vector for use in creating transgenic plants which carries a 6.7 kb fragment of the whole open reading frame (ORF) of the HAV, lacking the viral 5′UTR sequence, under the control of the plant patatin promoter and the omega (TMV) translation-enhancer box. A nos terminator is located 3′ to the HAV ORF. The vector carries the selection gene for kanamycin resistance (shown as the far left box, followed by the box that stands for the patatin promoter and omega enhancer, followed by the box that stands for the HAV ORF, and ending with the box on the far right, showing the nos terminator).

[0025]FIG. 1C shows the presence of HAV-specific RNA sequences in BY-2 cells transfected with pG35SΩHAV (a plasmid containing the entire HAV coding sequences under the control of the fruit-specific patatin promoter and the omega enhancer sequence) as determined by HAV specific polymerase chain reaction (PCR) techniques, using primers from the VP1-VP3 viral structural region of HAV: lanes 1-4 HAV-transfected cells; lane 5 negative control, and lane P positive control.

[0026]FIG. 2 depicts a dot blot and hybridization result with a radioactively labeled HAV-specific cDNA probe, which was used to detect HAV-specific sequences in DNA isolated from tomato fruits of 12 transgenic plant lines. As seen in the figure all the 12 plants contained integrated HAV DNA sequences.

[0027]FIG. 3 shows distribution of HAV-specific DNA sequences in the leafy parts of the transgenic plants as determined by HAV-specific PCR. M, molecular weight size marker; lanes 1-6, DNA from tomato leaves; P, positive control.

[0028]FIG. 4 shows radiolabeled HAV-specific dotblot cDNA probe analysis of RNA extracted from tomato fruits of 17 HAV transgenic tomato plants. To eliminate the possibility that positive signals might be due to residual DNA in the extracted plant tissue, the extracts were treated with DNase I and the dot blot analysis was repeated FIG. 4B). FIG. 4A; rows A1-A4, A6-A9, B1-B4, B6-B10, RNA from tomato fruits; D1 & D3, negative control; D10, positive control. FIG. 4B: A1-A9, B1B2, C1-C8, D1-D2, DNase-treated RNA extracts from tomato fruits; B-3, 100 pg HAV plasmid treated with DNase; B8 & D8, 100 and 200 pg, respectively of untreated HAV plasmid.

[0029]FIG. 5 shows Western blot analysis of HAV proteins from four different tomato lines that were earlier shown to express HAV RNA. Total proteins were extracted from tomatoes of lines 7-1, 12-1, 31-1 and 31-2 were subjected to Western blot analysis using a HAV-specific antibody raised to 70S HAV empty capsids. Results showed that HAV proteins were detectable at various levels in tomato fruits from all four transgenic plant lines.

[0030]FIG. 6 illustrates the experimental protocol used in two sets of experiments in which mice were fed with HAV transgenic or control tomato fruits to determine the potential immunogenicity of the plant-derived HAV vaccine.

[0031]FIG. 7 shows results of oral immunization of mice with transgenic tomato fruits. Balb/c mice were passively fed 5 times at weekly intervals with fruits from 4 different transgenic tomato lines, as indicated. Sera were tested for HAV-specific antibodies one week after the last feeding. The mice were subsequently boosted with 100 microliters of HAVRIX™ 1440 given intraperitoneally, 3 months after the first feeding with transgenic tomatoes. Sera were tested for HAV-specific antibody levels 3, 7 and 14 days later.

[0032]FIG. 8 shows the kinetics of primary and secondary anti-HAV antibody responses of Balb/c mice who were immunized with varying doses of HAVRIX™ 1440: 100 microliters, 10 microliters, 1 microliter and 0.1 microliters, Blood was collected at 3, 7 and 14 days after vaccination and serum titers of total HAV-specific antibodies were determined.

[0033]FIG. 9 shows the antibody response in mice after vaccination against HAV. The same groups of mice that were fed HAV transgenic tomatoes were immunized once with 50 microliters of BIO-HEP-B®(a commercial vaccine containing HbsAg), given intraperitoneally. Anti-HbsAg antibody levels in the serum were measured 7 and 14 days later.

[0034]FIG. 10 is a graph of the comparative serum titers measured in mice after administration of HAVRIX™ 1440 vaccine (containing 1440 ELISA Units (EU) of formalin-inactivated HAV adjuvanted with alum) by various routes. Mice (6/treatment group) received 100 microliters of HAVRIX™ by either the intranasal (IN), intrarectal (IR), oral (PO), intramuscular (IM), or intraperitoneal (IP) routes, twice on Days 0 and 21. Sera were collected 35 days after the first immunization and assessed for total HAV-specific antibody levels using the HAVAB EIA (Abbott Laboratories).

[0035]FIG. 11 illustrates the experimental protocol used for assessing the HAV-specific IgA response and cellular response to rectal or oral administration of unadjuvanted inactivated whole HAV antigen.

[0036]FIG. 12 shows the comparative frequencies of HAV-specific IgG antibody forming cells (AFC) observed in the spleens of mice that were immunized either intraperitoneally (IP) or intrarectally (IR) with varying doses of HAVRIX™ 1440 vaccine. Data shown in the figure represent the mean number of IgG-AFC determined by ELISPOT assays in spleen cell populations from 5 mice in each treatment group.

[0037]FIG. 13 shows the comparative frequencies of HAV-specific IgM antibody forming cells (AFC) observed in the lamina propria cell populations harvested from mice that were immunized either intraperitoneally (IP) or intrarectally (IR) with varying doses of HAVRIX™ 1440 vaccine. Data shown in the figure represent the mean number of IgM-AFC determined by ELISPOT assays in lamina propria cell populations from 5 mice in each treatment group.

[0038]FIG. 14 shows the comparative frequencies of HAV-specific IgA antibody forming cells (AFC) observed in the lamina propria cell populations harvested from mice that were immunized either intraperitoneally (IP) or intrarectally (IR) with varying doses of HAVRIX™ 1440 vaccine. Data shown in the figure represent the mean number of IgA-AFC determined by ELISPOT assays in lamina propria cell populations from 5 mice in each treatment group.

[0039]FIG. 15 shows the comparative maximal HAV-specific stimulation indices (SI) observed in the spleen cell (SPLC) populations harvested from mice that were immunized either intraperitoneally (IP) or intrarectally (IR) with varying doses of HAVRIX™ 1440 vaccine. SPLC were stimulated in vitro with concentrations of whole inactivated HAV varying from 20 to 2.5 EU/mL and lymphocyte proliferation was determined by uptake of radioactive DNA precursor (tritiated thymidine) after 7 days of culture. Data shown in the figure represent the mean of maximal stimulation index recorded from 5 mice in each treatment group.

[0040]FIG. 16 shows the comparative maximal HAV-specific stimulation indices (SI) observed in intestinal lamina propria lymphocyte (LPL) populations harvested from mice that were immunized either intraperitoneally (IP) or intrarectally (IR) with varying doses of HAVRIX™ 1440 vaccine. LPL were stimulated in vitro with concentrations of whole inactivated HAV varying from 20 to 2.5 EU/mL and lymphocyte proliferation was determined by uptake of radioactive DNA precursor (tritiated thymidine) after 7 days of culture. Data shown in the figure represent the mean of mammal stimulation index recorded from 5 mice in each treatment group.

[0041]FIG. 17 shows the comparative maximal HAV-specific stimulation indices (SI) observed in intestinal Peyer's patch cell (PPC) populations harvested from mice that were immunized either intraperitoneally (IP) or intrarectally (IR) with varying doses of HAVRIX™ 1440 vaccine. PPC were stimulated in vitro with concentrations of whole inactivated HAV varying from 20 to 2.5 EU/mL and lymphocyte proliferation was determined by uptake of radioactive DNA precursor (tritiated thymidine) after 7 days of culture. Data shown in the figure represent the mean of mammal stimulation index recorded from 5 mice in each treatment group.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] The present invention is of a vaccine produced in edible plant and/or animal products, as well as a method of producing a second-generation HAV vaccine through expression of at least one complete structure of a pathogen in a transgenic plant or animal, as well as by providing the vaccines themselves. Preferably, the present invention enables the production of virus-like particles in edible food plants, through the co-expression of a plurality of proteins and/or of a plurality of portions of such proteins. A “virus-like” particle is therefore herein defined as a group of co-expressed plurality of viral proteins and/or portions of such proteins. The co-expression of viral structural proteins should enhance the proper presentation of viral related antigens to the human immune system.

[0043] As a preferred example of the operation of the present invention, a vaccine was developed for hepatitis A virus (HAV). Previous attempts to vaccinate with isolated capsid proteins failed for HAV, because isolated HAV proteins were not sufficiently immunogenic to elicit neutralizing antibodies, which recognize specific conformational structures on the viral particle created only after the assembly of the capsid. To overcome this problem, preferably the present invention includes the construction of two HAV plasmids carrying a non-infectious HAV genome lacking the 5′UTR, for stable transformation of plants. In one plasmid (pGPPatΩHAV) transcription of HAV is driven from the patatin promoter for expression in tomato fruits, and in the second plasmid (pJDHAV) transcription is under the control of the 35SCaMV promoter and should be expressed in the green parts of transgenic plants. To assess the immunogenicity of the protein products of the HAV transgenes, the edible parts of transgenic plants were fed to experimental animals and their HAV-specific immune response was determined by laboratory tests.

[0044] According to preferred embodiments of the present invention, the technology which is developed for vaccines can also optionally and more preferably be applied for immunotherapeutic purposes, including but not limited to, oral immunization with chimeric virus like particles exhibiting specific tissue trophism to induce specific immunologic tolerance to pathogen antigens involved in inducing host tissue inflammation; or the use of chimeric virus like particles to target one or more genes, such as foreign or new genes for example, to specific organs, through the consumption of plant material containing non-infectious viral particles as gene vehicles.

[0045] To provide a comparison model system for oral administration of any type of vaccine against a pathogen that does not actually attack any portion of the digestive system, a method for orally administering a vaccine against HAV was developed. The development of an animal model for testing is described in Section 1. The method was first tested by using a traditional HAV vaccine (HAVRIX™) as described in greater detail below in Section 3. Next, transgenic plant products were fed to laboratory mice that served as a model mammalian system, as described in Section 2. Transgenic plants were constructed to express genes encoding proteins from the outer capsid of HAV, which served as the model pathogen. The HAV transgenic plants were fed to mice to determine if the HAV transgenes were expressing HAV capsid proteins in an immunogenic form and if these proteins were capable of eliciting HAV-specific antibody and cellular immune responses For comparison to conventional injection methods for vaccinating against HAV the immune response of the mice who were fed the HAV transgenic plant materials was compared to those elicited in other mice by a commercial HAV vaccine consisting of inactivated whole HAV adjuvanted with alum to enhance the immune response. The latter vaccine was administered to the mice by injection. These investigations are described in detail in Section 2, below.

[0046] To determine the utility of mucosal immunization via other routes such as the rectum or nasal cavity, as an alternative strategy to current methods of immunizing against HAV, conventional HAV vaccines containing alum-adjuvanted whole inactivated HAV, or whole inactivated HAV without alum, were employed for rectal, nasal, and oral immunization of mice. These investigations are described in detail in Section 3, below.

[0047] The present invention also optionally and preferably provides virus-like particles, which may also optionally be termed engineered viral particles, which are more preferably composed of a group of co-expressed plurality of viral proteins or portions thereof. Most preferably, the group of co-expressed plurality of viral proteins or portions thereof is expressed in an edible plant material. Hereinafter, the terms “engineered viral particles” and “virus-like particles” are used interchangeably. The use of such virus like particles, optionally and preferably in mucosal (oral, nasal or rectal) immunization may be used to induce specific immune tolerance to expressed viral antigens that are involved in chronic inflammatory diseases. Alternatively, this method may optionally be used to generate virus like particles encapsidating foreign or host genes as a gene delivery vehicle for gene therapy, that favors tissue specific targeting and permits prolonged tissue expression of the encapsidated gene. The induction of immune tolerance to the structural proteins of the delivery vehicle by oral immunization may optionally be exploited to enhance gene expression in target tissue.

[0048] Section 1: HAV as a Model Pathogen

[0049] The transgenic plant HAV vaccine was developed as a comparison model system for oral immunization against a pathogen that does not actually attack any portion of the digestive system but which uses the intestinal mucosal as a portal of entry in its infective process. The method of oral administration of a regular HAV vaccine is itself novel and non-obvious, as it is the first example of successful oral administration of such a vaccine, which is normally administered through intramuscular injection. Thus, the comparison model is also an inventive vaccine and method for administration thereof and is part of the present invention.

[0050] The gastrointestinal tract is a major port of entry for many pathogens (bacterial, viral, and parasitic) that impose a significant health burden. HAV, a positive-stranded RNA virus, is an example, HAV enters the human body through the gastrointestinal system, and migrates to the liver for tissue specific replication, thereby causing liver damage and clinical hepatitis.

[0051] To immunize the subject against the HAV disease, the comparison model of the present invention also includes mucosal (intrarectal) administration of conventional HAV vaccines, such as the currently available HAV vaccine, HAVRIX™ (SmithKline Beecham Biologicals).

[0052] The preferred vaccination strategy according to the present invention involves the exposure of the intestine to HAV particles either by oral administration or by topical application to the rectal mucosa, thereby potentially eliciting both an antibody response (optimally, neutralizing antibodies) and a cellular immune response as illustrated by data from the investigations described below.

[0053] Section 2. Construction of Transgenic Plants Expressing HAV Antigens and Their Use in Oral Immunization of Mice

[0054] Transgenic plants capable of expressing at least one complete, and appropriately assembled, HAV capsid structure were genetically engineered. According to this example, transgenic tomato plants were engineered to produce HAV viral particles that would be non-infectious because their genome excludes about 730 nucleotides of its 5′ NTR, without which, they can neither replicate nor express their own proteins. Elimination of this part of the viral genome does not affect the external capsid structure of the virus that is seen by the immune system, and therefore, by implication, the potential immunogenicity of the plant-produced HAV vaccine. Thus, when HAV transgenic tomatoes are eaten, the non-infectious viral particles with their immunogenic capsid proteins would be ingested as well. The plant-generated HAV virus-like particles should subsequently be taken up by M-cells on the surface of the gastrointestinal tract and transferred to antigen presenting cells and lymphocyte components of the mucosal immune system resulting in a HAV-specific response as illustrated by the data shown below.

[0055]FIG. 1A shows an example of the different types of virus-like particles that could optionally be produced in plants. On the right hand side of the figure, empty virus-like particles are shown, which could optionally be used for immunization against a particular virus as a vaccine for example. However, such particles could also optionally be used to induce tolerance, in order to actually specifically reduce the immune response to the particles, and hence to the viral capsid for example. For example, such tolerance could also optionally be used to reduce inflammation by reducing the immune response. Reduction of the immune response may optionally and preferably be desired in order to reduce the effects of diseases that have a viral component, preferably for inflammatory diseases that have a viral component. It should be noted that for some diseases, only particular variants that have a viral component may optionally be treated with the present invention.

[0056] A number of mechanisms of mucosal immunization, such as for rectal, nasal and targeted iliac lymph node immunization for example, have also been shown to induce non-cognate immune responses that reduce viral infection, for example for SIV (simian immunodeficiency various) and for SHIV (chimeric virus of SIV and SHIV). As the specific mechanisms involved would also influence the recruitment of inflammatory cells to tissues where the pathogen is established, they could also optionally be exploited to alter the inflammatory response and disease severity and progression (e.g., in chronic hepatitis C infection in the liver).

[0057] However, the use of empty viral particles is preferably not limited only to the treatment of diseases through either tolerance or immunization. Optionally, chimeric virus-like particles, that may for example include proteins from a plurality of viruses, may be used to induce both tolerance and immunity simultaneously. Such a combination is preferably determined according to the structure of the different proteins incorporated and/or their relative amounts. Chimeric virus-like particles may also optionally include one or more non-viral proteins.

[0058] The left hand side of the figure shows virus-like particles that encapsidate one or more genes, shown as a transgene. These virus-like particles may optionally be used to induce tolerance to the delivery vehicle, which may itself optionally be a virus containing the gene More preferably, empty virus-like particles are administered before the particles containing the transgene, in order to induce tolerance. Such tolerance is preferred in order to avoid inducing an immune response against the delivery vehicle of the transgene, and/or the transgene itself It should be noted that the capability to induce tolerance may be affected by a number of factors, including but not limited to, previous patient or subject exposure to one or more pathogens, and/or previous administration of one or more vaccines and/or other immunizing materials to the patient or subject. Therefore, optionally and most preferably, the virus-like particle contains proteins that are tailored to an individual patient and/or to a group of patients.

[0059] According to preferred embodiments of the present invention, selective tolerance to the antigens of a vector may also optionally be performed when the vector is a novel virus or a chimeric vector consisting of a xenovirus into which specific proteins have been incorporated that would enhance tissue targeting. For example, this vector could optionally be a gene therapy vector, for administering a transgene to a subject. Promoting tolerance to such a vector would be useful if the vector bad to be used on more than one occasion in the same subject, for example for repeated administration of the vector for gene therapy, as the initial induction of tolerance would obviate an immune response on secondary immunization. Thus, the vector could be used more than once without concerns of rejection.

[0060] All of these different effects are more preferably obtained by administering the plant material containing the virus-like particles to the subject, for example as food (oral administration) or through other mucosal administration, such as rectal and/or nasal immunization.

[0061] Depending upon the type of virus from which the proteins of the virus-like particles are taken, the virus-like particle may also optionally be targeted. For example, one or more HAV proteins, as described below, would preferably enable the virus-like particle to be targeted for the liver. One of ordinary skill in the art could easily select the proper protein(s) from the proper types of virus(es) for such targeting.

[0062] 2.1 Construction of Plasmid Vectors with the HAV Genome and Analysis of their Expression in Transgenic (Tg) Tomato Plant Tissues:

[0063] A 6.7 kb XbaI-PmeI HAV/7 cDNA fragment lacking the viral 5′UTR, and the additional 10 bp from the HAV translational start site, was isolated from plasmid pHAV/7. This fragment was inserted into the modified BSKS plasmid that contained the ten 5′ coding nucleotides of HAV/7. Further engineering inserted the 35SCaMV promoter and the Omega (TMV) translation-enhancer box in front of the HAV/7 coding region and a nos terminator behind this region. The promoter-HAV-terminator combination was finally cloned into a binary vector (for Agrobacterium tumefaciens-mediated genetic transformation) pGPTV-kan, termed pG35SΩHAV. This vector was introduced into Agrobacteria with a helper plasmid and BY-2 tobacco plant cells were transformed with pG35SΩHAV. Kan^(R) cell lines with were selected and established. The presence of HAV sequences in transfected BY-2 cells was confirmed by polymerase chain reaction amplification (PCR) using primers from the VP1-VP3 viral structural region of the virus (FIG. 1C).

[0064] A similar vector to the pG35SHAV was constructed replacing the 35.S CaUV promoter with the patatin promoter, which allows the transgene to be expressed in tomato fruits. This vector was termed pGPPATHAV (illustrated in FIG. 1B) was used to transform tomato plants by the Agrobacterium tumefaciens transformation procedure. It was previously assured that the patatin promoter, indeed, caused the expression of a marker gene (GUS) in the fruits of the transgenic plants. After genetic transformation of tomato tissue with this HAV construct (pGPPATHAV), 17 lines of transgenic (Tg) tomato plants were derived (46 plants in each line). These lines were assessed for the presence of HAV sequences in their genomic DNA. FIG. 2 depicts a dot blot and hybridization result with radiolabeled HAV cDNA probe of DNA extracted from tomato fruits of the Tg plant lines. As seen in the figure, all 12 lines contained HAV-specific DNA sequences confirming integration of the transgene. Some plant lines were also assessed by PCR for presence of HAV-specific sequences in DNA extracted from their leaves (FIG. 3)

[0065] Overall, DNA and RNA were extracted from 72 Tg plants, for the assessment of HAV integrated DNA sequences. In those plants showing integrated HAV DNA sequences, the presence of HAV RNA transcripts in nucleic acids extracted from the fruits was investigated. Total RNA extracted from Tg and control tomato plants was probed for HAV-specific RNA sequences by dot blot and hybridization assays FIG. 4A). To exclude the possibility that the positive signal seen was due to residual DNA in the RNA samples, the RNA extracted from the tomato fruits was treated with DNase I and the dot blot analysis was repeated (FIG. 4B). As seen in the figure; in some of the RNA samples, the specific signal was weaker after DNase I treatment (e.g. A1, A2, A5). However with other samples (A3, A5, A6, A8 and B1), the signal was observed to be higher than that of the control tomato (B2). Plants testing positive for HAV mRNA transcripts were further examined for expression of the inserted RNA. The predicted HAV protein products were detected by Western blot analysis with specific anti-HAV antibodies that included a mouse monoclonal anti-HAV (Biodesign International, Saco, Me., cat No. C65885M); a pooled antiserum produced from Balb/c mice immunized twice at 10-day intervals with 72 EU of HAVRIX™ vaccine administered intraperitoneally; and HAV-specific antibody raised to 70S HAV empty capsids. FIG. 5 shows a diagram of the observed expression of HAV proteins from four different tomato lines (7-1, 12-1, 31 -1 and 31-2), which had previously shown HAV RNA expression. Tomato tissue taken from all four Tg lines expressed HAV capsid proteins that were detected with the anti-HAV antibody directed to 70S empty capsids. Moreover, the HAV capsid proteins, VP1, VP2, and VP3 that together make up the conformational site to which virus-neutralizing antibodies are directed were detected in all four lines. Thus, the transgenic tomato plants not only express HAV proteins, but also more importantly, express those HAV capsid proteins relevant to the induction of a protective immune response.

[0066] 2.2. Evaluation of HAV Transgenic Plant Vaccines in Mice:

[0067] Two sets of feeding experiments employing similar protocols (shown in FIG. 6) were performed. Four groups of 4-5 week old female BALB/c mice (n=5, each) were fed tomatoes from Tg lines 7-1, 12-2, 31-1, and 31-2. Five control mice were fed with normal wild type tomatoes, such that they were exposed to the tomatoes continuously and fed ad libitum. In the second experiment separate test and control groups were fed the same tomatoes but were given 10 μg of cholera toxin (CT, Sigma-Aldrich) just prior to feeding. To ensure uptake, the mice were deprived of their standard chow for 12 hours before in the second experiment, such that exposure to the tomatoes was periodic and not continuous. The tomatoes were weighed, then left in the cages for 6 h, and then weighed again to estimate consumption. A total of 5 feedings were offered on Days 0, 7, 14, 21, and 28. Animals were bled on Day 35 for the preparation of sera for interim antibody testing. Sera from mice in each of the treatment groups were pooled for testing for total HAV-specific antibodies by a commercial inhibition ELISA (HAVAB EIA, Abbott Laboratories, Diagnostic Division, Abbott Park, Ill.).

[0068] In the first experiment, HAV-specific serum antibody was not observed in any of the treatment groups at the interim bleeding taken on Day 35 and thereafter (FIG. 7). To determine if oral administration had primed for a secondary response to HAV (in the absence of a detectable primary antibody response) the mice in all groups were subsequently boosted three months after the first feeding (indicated by the arrow in FIG. 7) by intraperitoneal (IP) injection with 100 μL of HAVRIX™ 1440 vaccine (an alum-adjuvanted commercial vaccine containing 1440 ELISA units [EU] of inactivated whole HAV) and the antibody response was followed by test bleedings taken on Days 95, 98, 102, and 109 as illustrated in FIG. 7. HAV-specific antibodies were detected after Day 98 in serum pools collected from the control, and Tg tomato groups 12-2, 31-1, and 31-2. Two groups of mice, the control group fed wild type tomatoes, and mice fed tomatoes from 31-1 Tg line, developed HAV-specific antibody responses against HAV with kinetics resembling a primary immune response. Two other groups (those fed Tg tomatoes from lines 12-2 and 31-2) demonstrated responses with slower kinetics. However, no antibody response was observed in mice that were fed Tg tomatoes from Line 7-1, as performed in the first experiment. Without wishing to be limited by a single hypothesis, the latter observation suggested that the antigen (or quantity of antigen) expressed by this Tg line might have induced oral tolerance to HAV antigens, a known possible outcome of oral immunization. The slower kinetics observed in the responses observed in mice that were fed Tg tomatoes from lines 12-2 and 31-2 also suggested some negative influence of priming by oral administration of HAV antigens. Hence, these results suggested that continuous exposure of the immune system of the mice to the HAV antigens appeared to induce immune tolerance instead of immunization against the virus. The usefulness of the induction of tolerance was described above, and is also addressed in greater detail below, in Sections 3 and 4.

[0069] To establish whether oral immunization with the transgenic HAV antigen had, indeed, induced specific immune tolerance to HAV a series of experiments were conducted. First, the kinetics of the primary and secondary antibody response to administration of the HAV vaccine were characterized by immunizing Balb/c mice with HAVRIX™ 1440 (FIG. 8). The commercially available HAVRIX™ vaccine is also known as HAVRIX™ 1440 vaccine, and is a sterile suspension containing formaldehyde-inactivated hepatitis A virus (HM175 hepatitis A virus strain) adsorbed onto aluminium hydroxide. The virus was propagated in MRC5 human diploid cells. Before viral extraction the cells were extensively washed to remove culture medium components. A virus suspension was then obtained by lysis of the cells, followed by purification using ultrafiltration techniques and gel chromatography. The virus was inactivated with formalin. The vaccine contained 1440 E.U. (Elisa units) of viral antigens, in a 1.0 ml dose volume. The inactive ingredients in the vaccine were aluminum (from aluminium hydroxide), 0.25 mg; amino acids for injection, 1.50 mg; disodium phosphate, 0.575 mg; monopotassium phosphate, 0.100 mg; sodium chloride, 4.500 mg; potassium chloride, 0.115 mg; polysorbate 20, 0.025 mg; 2-phenoxyethanol, 2.500 mg, neomycine sulfate, 10 ng; formaldehyde, 100 micrograms; water, 0.5 ml; all contained in a 1.0 ml dose volume. The vaccine was obtained from SmithKline Beecham Biologicals S.A. (Belgium). Each group of 5 mice received a different dose of vaccine, as indicated. After 3, 7 and 14 days, the mice were bled and sera were tested for anti-HAV antibodies.

[0070] Mice receiving 100 microliters of vaccine developed a typical primary antibody response: 7 days after the vaccination, HAV-specific antibodies were detected at low levels and remained unchanged for 14 days (FIG. 8). HAV-specific antibodies were not detected in mice receiving the lower amounts of HAVRIX™ 1440 (10, 1 and 0.1 microliters) over the period of evaluation. To determine if the lower doses of HAVRIX™ primed immunologic memory in the absence of a detectable primary antibody response, the mice were boosted with 100 microliters of HAVRIX™ 1440 one month after the first vaccination (indicated by the arrow in FIG. 8) to determine whether a secondary antibody response could be elicited. Presence of serum HAV-specific antibodies was checked 3, 7 and 14 days after the vaccination. As seen in FIG. 8, in the group of mice that developed detectable primary antibody response, a brisk secondary anti HAV specific antibody response was found, with rapid kinetics (the antibodies were detectable as early as 3 days after boosting) and these reached a higher level than in mice primed with the lower doses of HAVRIX™. Mice in the groups that were primed with lower doses of HAVRIX™, also demonstrated a HAV-specific antibody response but with slower kinetics compared to that of the group who received the highest priming dose of HAVRIX™. These results showed that low doses of HAVRIX™ given intraperitoneally could prime for immunologic memory (in the absence of a detectable primary antibody response) and that this memory was sufficient to result in a detectable antibody response upon challenge with HAVRIX™. Alternatively, the response observed in those animals that were originally immunized with the lower doses of HAVRIX™, then subsequently re-immunized with 100 microliters of HAVRIX™ 1440, may have represented a true primary immune response to the vaccine. In order to show that the inhibition of the immune response observed in the initial feeding experiment with HAV Tg tomatoes was specific to the HAV antigen, the mice from that experiment were immunized with an irrelevant antigen, the HbsAg of the hepatitis B virus using a commercial HAV vaccine preparation (BIO-HEP-B®, Bio-Technology Israel). Mice were immunized once with BIO-HEP-B® and HbsAg-specific antibody levels were measured 7 and 14 days after the administration of the vaccine. As shown in FIG. 9, mice in all treatment groups developed a specific HbsAg antibody response indicating that oral administration of Tg HAV antigens had not induced a generalized state of immunologic nonresponsiveness.

[0071] In the second experiment, again all animals proved to be HAV seronegative after administration of Tg HAV vaccines. The second group which were co-administered cholera toxin (CT), a mucosal adjuvant shown to enhance the immune response to other antigens given orally, also did not develop measurable immunity after feeding with Tg tomatoes. All mice were subsequently given a subimmunogenic dose (72 EU) of HAVRIX™, intraperitoneally (IP), one week later. Two to three weeks later, animals were killed and their spleens were removed for analysis of HAV-specific antibody forming cell (AFC) numbers by ELISPOT assay and in vitro lymphocyte proliferative responses by lymphocyte stimulation tests (LST)(methods described in Section 3). Sera were prepared for antibody testing. In marked contrast to the results of the first experiment, feeding with Tg tomatoes from Line 7-1 appeared to have stimulated a HAV-specific memory response. This resulted in enhanced HAV-specific antibody and cellular responses to a subsequent IP injection of a sub-immunogenic dose of HAVRIX™ vaccine relative to control animals that were fed normal tomatoes (Tables 1 and 2, below). This type of response was not observed with the other Tg tomato lines that were evaluated.

[0072] Conclusions: Therefore, while the data from the first set of feeding experiments with HAV transgenic tomatoes suggested the possibility of induction of specific oral tolerance (to HAV antigens), which without wishing to be limited to a single hypothesis, may be due to the continuous exposure of the mice to the transgenic tomatoes, data from the second set of experiments revealed that the ingested transgenic HAV antigen could also prime for HAV-specific immunologic memory allowing for a strong secondary response upon parenteral challenge. It may also be possible that differences in the amounts of transgenic HAV antigen ingested by the animals between the two experiments account for these different outcomes, again without wishing to be limited by a single hypothesis. TABLE 1 Immune responses of animals passively fed with HAV-Tg tomato line 7-1 or control (normal) tomatoes after boosting with 72 EU of HAVRIX ™ given intraperitoneally. Max. SI Animal No. of AFC/10⁶ SPLC^(a) Attain- Total HAV- No. Treatment IgG IgM IgA ed^(b) Specific Ab^(c) 7-1A 7-1 alone 0 4 0 3.5 0 7-1B 7-1 alone 6 38 0 7.5 15 7-1C 7-1 alone 10 68 0 6.0 21 7-1/6 7-1 alone 2.1 25.4 1.7 4.2 20 C37 Control 0 5 0 2.7 0 C39 Control 0 11.6 2 2.1 22 C40 Control 0 45 0 2.1 4 C42 Control 0 0.5 0 1.1 14 7-1/7 7-1 + CT 4.2 15.8 0 4.6 19 7-1/8 7-1 + CT 0 15.8 0.8 5.7 21 7-1/9 7-1 + CT 0 14.2 0.4 8.1 0 7-1/10 7-1 + CT 10.4 10.4 0 9.4 4 7-1/11 7-1 + CT 0.4 3.75 0.8 8.1 20 7-1/12 7-1 + CT 0 9.2 0 6.2 19 C38 Control + CT 0 0.4 0.4 1.8 0 C41 Control + CT 0 24.6 0.8 3.0 11 C43 Control + CT 0 4.6 0 2.7 2 C44 Control + CT 0.8 10.4 0.8 7.4 0 C45 Control + CT 1.25 12.5 0 3.4 14

[0073] TABLE 2 Comparison of medians (calculated from data shown in TABLE 1) by treatment. No. of AFC/10⁶ cells Max. SI Total HAV Treatment IgG IgM IgA Attained Specific Ab 7-1 alone 4.1 31.7 0 5.1 17.5 Control 0 11.6 0 2.1 4.0 7-1 + CT 0.2 12.3 0.2 7.2 19.0 Control + CT 0 10.4 0.4 3.0 2.0

[0074] Section 3: Rectal Immunization of Mice with Whole HAV Vaccines

[0075] In order to test the second method of the present invention, an animal model was developed for the assessment of the mucosal and systemic immune responses to whole HAV following rectal administration of formalin-inactivated whole virus either in the form of unadjuvanted semi-purified HAV prepared from FRhK4 cells or as the commercial vaccine, HAVRIX™ consisting of highly purified whole HAV, inactivated and adsorbed with alum to enhance its immunogenicity. A series of experiments were conducted and are described in chronological order below.

[0076] 3.1 Preliminary Investigations of the Comparative Immune Response to Delivery of HAVRIX™ via Mucosal and Conventional Systemic Routes:

[0077] In the first experiment, female Balb/c mice, aged 4-6 weeks, were injected with varying amounts of HAVRIX™ at a concentration of 720 EU/mL, using three different routes and doses. 100 microliters (72 EU) intraperitoneally (IP); 100 microliters (72 EU) intrarectally (IR); or 20 microliters (14.4 EU) intranasally (IN). The vaccine was administered on Days 0 and 21. In a companion experiment using a higher-titered HAV vaccine, HAVRIX™ 1440, mice (6/treatment group) received 100 microliters (144 EU) by either the intranasal (IN), intrarectal (IR), oral (PO), intramuscular (A, or intraperitoneal A) routes, twice on Days 0 and 21. In both experiments sera were collected from the mice 35 days after the first immunization and were then tested for total specific HAV antibodies using a commercial kit (HAVAB, Abbott Laboratories), Cellular responses to HAV were evaluated by three methods: the lymphocyte stimulation test which evaluated lymphocyte proliferation to in vitro stimulation with HAV virus; HAV-specific assays for detection of cytotoxic T lymphocytes (CTLs) from their ability to mediated in vivo killing of target cells expressing HAV antigens; and the in vivo delayed hypersensitivity test (footpad swelling in response to injection of HAV virus). The results of the first experiment using the lower titered HAVRIX™ 720 formulation and variable doses revealed that mice receiving the vaccine through the IP (conventional route) or IR routes, developed anti-HAV antibodies, whereas mice that received the vaccine intranasally, did not (data not shown). The lower titer HAVRIX™ 720 formulation contains the same ingredients as described above for the HAVRIX™ 1400 formulation, except that the former only contains 720 E.U. of HAV antigens. The vaccine was obtained from the same source as the HAVRIX™ 1400 formulation, as described above. The results of the second experiment, shown in FIG. 10 were similar, showing that all the mice that received the vaccine IP or IM, while only about 50% of the mice that received the vaccine IR, developed anti-HAV antibodies. The response after IP or IM vaccination was higher than after immunization by the mucosal routes (IR, PO, IN). Mice that were vaccinated intranasally or orally developed barely detectable levels of anti-HAV antibodies. Evidence of a cellular immune response in the HAV seropositive mice was sought. Specific responses to in vitro stimulation with HAV in proliferation assays could not be detected in any of the treatment groups. Similarly, no CTL response was observed in cytotoxicity assays, likely due to the presence of alum adjuvant that is known to suppress CTL activity. In delayed type hypersensitivity experiments, HAVRIX™ or PBS as control was injected to the footpad of the vaccinated mice, and swelling was measured after 24 and 48 hours. Regardless of the immunization route, most of the mice showed a marked reaction after HAVRIX™ injection but not after PBS injection (data not shown).

[0078] 3.2 Investigations of Systemic and Mucosal Immune Responses to Inactivated Whole HAV:

[0079] As the results of preliminary experiments with HAVRIX™, an alum-adjuvanted whole HAV vaccine suggested low immunogenicity of the vaccine when it was administered by mucosal (IR, IN, PO) routes in comparison with systemic (IP, IM) routes, a series of experiments were conducted to ascertain whether the presence of alum adjuvant, or dosage levels, influenced the responses observed in the preliminary experiments. These experiments were also designed to develop or implement the methodology required to assess intestinal mucosal immune responses. In these experiments, only the IR or PO routes of immunization were used, as these were considered relevant to the infectivity and pathogenicity of HAV.

[0080] 3.2.1 Immunization of Mice with Whole HAV Without Alum:

[0081] The first series of experiments addressed the issue of alum adjuvant as a potential inhibitor of the mucosal immune response to HAV. For these experiments, a semi-purified preparation of HAV grown on FRhK4 monkey kidney cells and inactivated with formatin was used as a rectal, oral, or intraperitoneally delivered immunogen. Transgenic tomato HAV antigen was also used as an oral immunogen. The experimental protocol is illustrated in FIG. 11. Female (non-SPF) Balb/c mice, aged 4-6 weeks at the outset of experiments were used and kept under standard non-SPF conditions. Specific antigens employed were: semi-purified formalin-inactivated HAV (strain HM-175 grown in FRhK4 cells, as in HAVRIX™ vaccine) purchased from Microbix Biosystems, Inc, Toronto, Canada (HAV Grade 2 antigen, cat. No EL 25-02), that according to the manufacturer, contained 2.2 mg/mL of protein and approx. 6.0 EU/mL of HAV. This antigen was used to immunize mice in two different presentation formats. For intraperitoneal (IP) immunization, the antigen was emulsified 1:1 with incomplete Freund's adjuvant (IFA). For immunization via the intrarectal (IR) or oral (PO) routes, the antigen was diluted 1:1 with sterile saline and delivered with a No. 1 surgical catheter attached to a tuberculin syringe. Oral antigen was offered passively and invasive gavage was not employed. For all immunizations in this series of experiments dosage volume was 100 microliters is (containing approx 110 micrograms of total protein or 0.3 E.U. of HAV).

[0082] Transgenic plant HAV vaccines produced by two tomato strains (2-21 and 3-12), both of which were shown to express mRNA for the HAV insert, were also evaluated. For immunization, approx. 0.4 g of lyophilized transgenic plant material was suspended in 4 mL of sterile saline and sonicated on ice for 3 min using 10-second bursts. The resultant material was centrifuged and the supernatant used to immunize mice, giving each animal 100 microliters PO. Sterile saline either emulsified with IFA (for i.p. injections) or given alone (for IR or PO routes) served as a negative control.

[0083] The immunization protocol, schedule and numbers of animals used were as follows. Animals immunized via the IP or IR routes received two doses at 12-day intervals while animals immunized orally received 4 doses at 5-day intervals. Three weeks after the final immunizations approx. 0.5 mL of blood was obtained from the tail vein of each mouse. To determine which mice were likely to have responded to immunization sera were prepared and tested using a commercial competitive inhibition enzyme immunoassay (ETI-AB-HAVK-3 [anti-HAV], Sorin BioMedica, Italy) designed for measuring total HAV-specific antibody in human serum with a sensitivity of <20 WHO mIU/mL As the assay is a competitive inhibition method, in which the antibody to be tested competitively inhibits binding of HAV-specific antibody provided with the kit, the assay was used to determine if any of the mice had produced HAV-specific IgG capable of inhibiting the binding of the assay reagent. While none of the animals tested was definitively positive for HAV-specific IgG by this method, two of the mice immunized with HAV/IFA via the IP route had borderline levels of specific IgG.

[0084] As these negative results did not preclude the possibility that the mice may have responded in the mucosal immune compartment, the animals were subsequently randomized among the treatments and tested in two groups. One group was killed in subgroups of 6-8 mice over a period spanning 7-28 days after the last immunization. Spleens and Peyer's patches were harvested and cell populations were prepared for stimulation in vitro with HAV or control antigens (see below) and evaluation of the HAV-specific IgA response using ELISPOT and EIA, and HAV-specific proliferation indices as described in detail below. The second group of mice was allowed to rest an additional three weeks, and then they received a single intradermal (ID) boost of HAV antigen (27.5 μg of total protein or 0.6 EU). These mice were killed as groups of 6-8 over a period of 7-21 days. This latter group was designated as “in vivo boosted” in contrast to the former group whose cells were restimulated with HAV antigen in vitro.

[0085] Preparation of Serum, Spleen Cell and Peyer's Patch Cell Populations:

[0086] Animals were killed by an overdose of chloral hydrate. Blood was collected by cardiac puncture, allowed to clot overnight at 4 C. Serum was prepared and kept frozen at −20° C. until tested. The spleen and Peyer's patches (approx. 4-8 per animal) were removed into sterile RPMI-1640 medium (GIBCO/BRL, Biological Industries, Beit Haemek, Israel) containing antibiotics (penicillin, 100 U/mL and streptomycin, 100 micrograms per mL, GIBCO/BRL) and transported to the laboratory for further processing. The organs were washed once in sterile RPMI. Spleen cell (SPLC) suspensions were prepared by teasing apart the spleen in 5 mL of RPMI+antibiotics using sterile forceps. The suspension was transferred into a sterile conical bottomed 15 mL tube and the tissue debris allowed to settle. The supernatant containing suspended SPLC was transferred into a fresh tube and the cells were pelleted by centrifugation (1500 rpm, 7 min) at room temperature. The cells were washed twice by centrifugation (1500 rpm, 5 min) through RPMI+antibiotics then viable cell numbers were determined by Trypan Blue exclusion using a Neubauer hemocytometer. The SPLC were then resuspended in 10 mL of the same medium containing 10% fetal calf serum (FCS, HyClone Laboratories, Tarom Applied Technologies, Petah Tikvah, Israel) at a concentration of 1×10⁶ cells/mL for use in subsequent assays. Similarly, Peyer's patches were teased apart in 5 mL of RPMI+antibiotics containing 1.5 mg/mL of Dispase (Sigma-Aldrich, Milwaukee, Wis.). The Peyer's patch cell (PPC) suspension was transferred into a conical-bottomed 15 mL tube and incubated with shaking for 45-60 min at 37° C. to dissociate lymphoid cells from the tissue stroma. The PPC were then pelleted by centrifugation and washed twice through RPMI+antibiotics and numbers of viable cells were determined as described above. As PPC numbers were usually limited, the final suspensions in 4.5 mL of RPMI containing antibiotics and 10% FCS, whose densities ranged from 1×10⁴ to 5×10⁵ cells/mL, were used in assays without further adjustment.

[0087] Determination of Capacity of SPLC and PPC to Synthesize and Secrete HAV-Specific IgA Antibodies in vitro and Determination of Proliferative Response to HAV Antigens:

[0088] After determination of cell densities, SPLC and PPC preparations from each mouse were pipetted in 1-mL aliquots into 5 wells of flat-bottomed sterile 24-well tissue culture plates. For each cell population, separate wells received: medium only (unstimulated negative control); pokeweed mitogen (Sigma) at a final concentration of 10 micrograms per mL and HAV antigen (Microbix) diluted to give final concentrations per well of 11, 5.5, 2.8, and 1.4 micrograms per mL. The plates were incubated for 5 days at 37° C., 5% CO₂ in air. At the end of this culture period, 0.7 mL of supernatant was removed from each well and stored at −20° C. for determination of HAV-specific IgA by enzyme immunoassay (EIA, see below). For assays in which HAV-specific IgA forming cells were evaluated after in vitro stimulation the cells were treated as follows. Cells from each well were harvested into conical-bottomed tubes, washed once by centrifugation through RPMI+antibiotics and resuspended in 1 ml of RPMI containing antibiotics and 10% FCS. Viable cell numbers after in vitro stimulation were determined by Trypan Blue exclusion and related to cell densities in the initial cell population to give a stimulation index (SI). SI's≧2.0 were considered a positive response to stimulation.

[0089] ELISPOT Assays to Determine Frequencies of IgA Antibody Forming Cells (AFC) in Systemic (SPLC) and Mucosal (PPC) Immune Compartments:

[0090] To detect cells producing specific IgA antibodies in both the spleen and Peyer's patches, an ELISPOT assay was adapted from a standard protocol Lewis D J M & Hayward C M M, Stimulation of Mucosal Immunity. In: Vaccine Protocols; A. Robinson, G H Farrar, C N Wiblin, Eds., Humana Press, Totowa, N.J., 1996, pp 187-195). The principle of this assay is that cells forming specific IgA will secrete it onto the surface of antigen-coated microplate wells, leaving an “imprint” which may be detected by conventional enzyme-linked immunosorbent assay (ELISA) techniques. As nonspecific background reactivity is inherent in the ELISPOT method, all assays contained controls for reactivity with FRhK4 antigens (present in the original immunogen) and nonspecific binding to other assay constituents (coating buffer control). Wells of 96-well polystyrene tissue culture plates (Costar 3596, Corning Inc., Corning, N.Y.) were coated overnight with HAV antigen (Microbix), or FRhK4 lysate, (prepared by freeze thawing uninfected cells and clarifying the supernatant by centrifugation at 15,000 rpm for 20 min), diluted to 20 micrograms per mL of protein in standard ELISA coating buffer (HCO₂/CO₃ ²⁻, pH 9.6). Negative control wells received coating buffer only. Coating volume was 100 microliters per well and duplicate wells were coated for each antigen or control.

[0091] The following day the antigens were discarded and the wells were washed 4 times with sterile phosphate buffered saline (PBS, pH 7.4). The wells were subsequently blocked for 2-4 h at room temperature with 5% FCS in sterile PBS (250 microliters per well). The blocking solution was discarded and SPLC or PPC suspensions were added to the wells and the plates were incubated for 24 h at 37° C. in 5% CO₂ in air, taking care to keep the plates level and undisturbed during this period. SPLC were incubated at a density of 100,000 cells/well in a volume of 100 microliters per well while PPC were used at densities of 1000-50,000 cells/well (100 microliters per well) according to their yields. At the end of the incubation period the cells were discarded and the microplate wells were washed 5 times in PBS containing 0.05% Tween 20 (PBST). To detect spots where antigen-specific IgA-forming cells (IgA-AFC) had secreted their antibodies into the antigen-coated microplate wells, goat-anti-mouse IgA (affinity purified from Kirkegaard & Perry Labs [KPL], Gaithersburg, Md.) diluted 1:500 in PBST containing 5% normal rabbit serum (NRS, Biological Industries, Beit Haemek, Israel), 100 microliters per well was added and the microplates were incubated overnight at 4° C. The antibody was discarded and the wells were washed 5 times with PBST. Next, alkaline phosphatase-conjugated rabbit anti-goat IgG (affinity-purified, KPL) diluted to 1:1000 in PBST containing 5% NRS, 100 microliters per well, was added and the microplates were incubated for 2 h at room temperature. The antibody solution was discarded and the microplates were washed 5 times with PBST. To detect spots representing single AFC, alkaline phosphatase substrate (BCIP/NBT SigmaFast, Sigma) prepared according to the manufacturer's directions, was added (100 microliters per well), and the enzymatic reaction was allowed to proceed at room temperature in the dark for 45 min before stopping it by rinsing the plates 3 times in distilled water. The plates were covered with aluminum foil and stored at 4° C. until AFC counting. Spots were enumerated using an inverted microscope with 400× magnification. Spots in duplicate wells for HAV and control antigens were counted and averaged. To calculate the net number of HAV-specific AFCs, the number of spots in counted the control wells were averaged then this average was subtracted from the averaged number of spots calculated in wells coated with HAV antigen. The net HAV-specific spot (AFC) number was reported (Tables 3-8, below).

[0092] EIA for Detection of HAV-Specific Antibodies in Mouse Serum and Tissue Culture Supernatants:

[0093] During the course of these experiments, preliminary development of an enzyme immunoassay (EIA) for detecting HAV-specific IgA antibodies in mouse serum or in tissue culture supernatants was undertaken. Wells of polystyrene EIA microplates (Nunc Immunopolysorp, Fisher Scientific, Israel) were coated overnight at 4° C. with HAV antigen (Microbix) or FRhK4 antigen, diluted to 20 micrograms per mL of protein in HCO₂ ⁻/CO₃ ²⁻, pH 9.6 coating buffer as described above (100 microliters per well, in duplicates). Negative control wells received coating buffer only. The antigen or control solutions were discarded and the wells were washed three times in PBS containing 0.05% Tween 20 (PBST). Wells were subsequently blocked for 1 h at room temperature with 250 microliters of blocking buffer (5% NRS in PBST). The blocking solution was discarded and the wells were washed 5 times with PBST. Tissue culture fluids to be tested for in vitro production of HAV-specific IgA or dilutions of mouse serum from immunized animals (1:20 and 1:200 in blocking buffer) were subsequently added (100 microliters per well, single determinations for each antigen and negative control) and the plates were incubated overnight at 4° C. These solutions were discarded and the wells washed 5 times in PBST as before. Next, goat anti-mouse IgA (KPL) diluted to 1:500 in blocking buffer (100 microliters per well) was added and the plates were incubated for 2 h at room temperature. The wells were emptied and washed 5 times with PBST. Alkaline phosphatase (AP)-conjugated goat anti-rabbit IgG (KPL, 1:1000 dilution in blocking buffer, 100 microliters per well) was added and the plates were incubated for a further 2 h at room temperature. After discarding the AP conjugate and washing the wells 5 times with PBST, AP substrate (SigmaFast NBT substrate, Sigma, prepared according to the manufacturer's directions was added and the plate was incubated for 1 h at room temperature. Absorbance at 405 nm (A₄₀₅) determined using an EIA microplate reader (Tecan Spectra Rainbow). For each animal and treatment, results were expressed as a signal-to-noise (S/N) ratio obtained by dividing the A₄₀₅ determined with antigen (either HAV or FRhK4) by the A₄₀₅ recorded in wells receiving coating buffer only. S/N ratios≧2.0 were considered positive.

[0094] Lymphocyte Stimulation Assays:

[0095] To assess lymphocyte recognition of HAV antigens in both the systemic (represented by SPLC) and gut mucosal (represented by PPC) immune compartments, in vitro lymphocyte proliferation assays were performed. Proliferation assays were performed as follows for experiments where the response to in vivo boosting with HAV was evaluated. SPLC and PPC (when in sufficient quantity) were adjusted to 1×10⁶ cells/mL in RPMI containing antibiotics and 10% FCS. Proliferation assays were performed in flat-bottomed 96-well tissue culture plates. Each well received 100 microliters of cell suspension, 100 microliters of medium (as above), and 10 microliters of one of the following: medium only (unstimulated control); phytohemagglutinin (Sigma) prepared to give a final well concentration of 10 micrograms per mL; or HAV antigen (Microbix) diluted to give final well concentrations of 11, 5.5, 2.8, and 1.4 micrograms per mL. All concentrations are expressed as micrograms of protein/mL. Duplicate wells were set up for each stimulant. The plates were incubated for 5 days at 37° C. in 5% CO₂ in air. To determine the extent of cellular proliferation, 2 μCi of ³H thymidine (Amersham, Israel) was added to each well during the last 18-24 hours of culture. The cells were harvested by hypotonic lysis and their DNA collected onto glass fiber filters for determination of incorporated radioactivity by liquid scintillation counting. Stimulation indices (SI) were determined by dividing the averaged (over duplicates) cpm obtained for the stimulated wells by the averaged cpm obtained for the negative control (medium only) wells. SI's≧2.0 were considered positive. TABLE 3 Results of Bulk Culture Experiments: Mice immunized with HAV intraperitoneally (IP). LST EIA ELISPOT Immunization Stimulation Specific IgA No.IgA-AFC Protocol/ Index (SI)^(b) (S/N)^(c) (Mean^(d)) Stimulant^(a) SPLC PPC SPLC PPC SPLC PPC HAV/IFA IP #1: None — — 1.7 2.1 0 0 Mitogen (10 μg/mL)^(e) 0.5 1.9 1.7 1.8 1.5 0 HAV (11 μg/mL) 0.5 2.3 2.3 2.0 10.1 1.5 HAV (5.5 μg/mL) 0.5 0.8 1.7 1.5 17 0 HAV (2.8 μg/mL) 0.7 2.6 1.8 1.6 0 0 HAV (1.4 μg/mL) ND ND ND ND ND ND HAV/IFA IP #2: None — — 1.5 1.4 5 0 Mitogen (10 μg/mL)^(e) 1.3 1.6 1.5 1.0 0 0 HAV (11 μg/mL) 2.3 1.3 1.6 1.6 25.5 0.5 HAV (5.5 μg/mL) 1.5 2.2 1.5 1.3 11.2 0 HAV (2.8 μg/mL) 1.0 1.9 2.0 1.6 21.7 0 HAV (1.4 μg/mL) ND ND ND ND ND ND

[0096] TABLE 4 Results of Bulk Culture Experiments: Mucosally-immunized mice. LST EIA ELISPOT Immunization Stimulation Specific IgA No.IgA-AFC Protocol/ Index (SI)^(b) (S/N)^(c) (Mean^(d)) Stimulant^(a) SPLC PPC SPLC PPC SPLC PPC HAV IR #1: None — — 1.4 1.1 0 0 Mitogen (10 μg/mL)^(e) 1.1 0.3 1.3 2.3 0 0 HAV (11 μg/mL) 1.2 0.6 1.6 1.8 43 0 HAV (5.5 μg/mL) 1.0 1.2 1.4 1.7 31.3 0 HAV (2.8 μg/mL) 2.2 1.3 1.4 1.5 16.5 0 HAY (1.4 μg/mL) ND ND ND ND ND ND HAV IR #2: None — — 1.3 1.5 65.7 0 Mitogen (10 μg/mL)^(e) 0.1 3.0 1.1 1.6 0 0 HAV (11 μg/mL) 1.3 8.0 1.4 1.5 20.2 0 HAV (5.5 μg/mL) 1.0 5.4 1.5 1.7 39 0 HAV (2.8 μg/mL) 1.0 6.1 1.4 2.2 55.2 0 HAV (1.4 μg/mL) ND ND ND ND ND ND HAV IR #3: None — — 1.0 1.1 0 0 Mitogen (10 μg/mL)^(e) 0.1 2.0 0.9 1.9 0 0 HAV (11 μg/mL) 1.1 5.0 0.9 1.3 0 0 HAV (5.5 μg/mL) 1.2 3.0 2.0 1.6 0 0 HAV (2.8 μg/mL) 0.4 3.9 1.1 1.7 17 0 HAV (1.4 μg/mL) ND ND ND ND ND ND HAV IR #4: None — — 1.0 1.0 1.5 0 Mitogen (10 μg/mL)^(e) 1.1 1.7 1.3 1.4 0 0 HAV (11 μg/mL) ND ND ND ND ND ND HAV (5.5 μg/mL) 1.2 2.4 1.4 1.4 2 0 HAV (2.8 μg/mL) ND ND ND ND ND ND HAV (1.4 μg/mL) ND ND ND ND ND ND HAV IR #5: None — — 1.4 1.5 0.5 0 Mitogen (10 μg/mL)^(e) 0.8 1.4 1.3 1.4 0 0 HAV (11 μg/mL) ND ND ND ND ND ND HAV (5.5 μg/mL) 1.1 1.6 1.4 1.4 0 0 HAV (2.8 μg/mL) ND ND ND ND ND ND HAV (1.4 μg/mL) ND ND ND ND ND ND HAV PO: None — — 1.4 1.5 24 0 Mitogen (10 μg/mL)^(e) 0.4 4.2 1.6 0.9 0 0 HAV (11 μg/mL) ND ND ND ND ND ND HAV (5.5 μg/mL) 1.2 1.7 1.5 1.4 17 0 HAV (2.8 μg/mL) ND ND ND ND ND ND HAV (1.4 μg/mL) ND ND ND ND ND ND

[0097] TABLE 5 Results of Bulk Culture Experiments: Negative Controls LST EIA ELISPOT Immunization Stimulation Specific IgA No.IgA-AFC Protocol/ Index (SI)^(b) (S/N)^(c) (Mean^(d)) Stimulant^(a) SPLC PPC SPLC PPC SPLC PPC Saline/IFA IP: None — — 1.8 1.5 0 0 Mitogen (10 μg/mL)^(e) 0.3 1.0 1.7 1.5 0 0 HAV (11 μg/mL) 1.6 1.1 1.7 1.5 4.2 0 HAV (5.5 μg/mL) 0.6 2.0 1.6 1.4 1.8 0 HAV (2.8 μg/mL) 0.8 1.2 1.5 1.4 0 0 HAV (1.4 μg/mL) ND ND ND ND ND ND Saline PO: None — — 1.4 1.5 0 0 Mitogen (10 μg/mL)^(e) 1.0 2.3 1.6 0.9 0 0 HAV (11 μg/mL) ND ND ND ND ND ND HAV (5.5 μg/mL) 0.9 1.3 1.5 1.4 0 0 HAV (2.8 μg/mL) ND ND ND ND ND ND HAV (1.4 μg/mL) ND ND ND ND ND ND

[0098] TABLE 6 Results of Experiments Following Boosting of Immunity to HAV in mice primed via the IP route. LST EIA ELISPOT Immunization Stimulation Specific IgA No.IgA-AFC Protocol/ Index (SI)^(b) (S/N)^(c) (Mean^(d)) Stimulant^(a) SPLC PPC SPLC PPC SPLC PPC HAV/IFA IP #1: None — — 4.4 ND 12.3 2.5 Mitogen (10 μg/mL) 2.6 1.4 3.0 ND ND ND HAV (11 μg/mL) 2.0 0.8 2.5 ND ND ND HAV (5.5 μg/mL) 0.7 1.2 2.5 ND ND ND HAV (2.8 μg/mL) 0.9 1.1 3.5 ND ND ND HAV (1.4 μg/mL) 1.2 1.8 2.2 ND ND ND HAV/IFA IP #2: None — — 3.2 ND 0 7.5 Mitogen (10 μg/mL)^(e) 1.3 2.8 2.2 ND ND ND HAV (11 μg/mL) 0.9 2.4 1.7 ND ND ND HAV (5.5 μg/mL) 1.4 3.0 1.7 ND ND ND HAV (2.8 μg/mL) 1.1 2.7 1 6 ND ND ND HAV (1.4 μg/mL) 1.2 4.2 1.6 ND ND ND

[0099] TABLE 7 Results of Experiments Following Boosting of Immunity to HAV: Mucosally-Immunized Mice. LST EIA ELISPOT Immunization Stimulation Specific IgA No.IgA-AFC Protocol/ Index (SI)^(b) (S/N)^(c) (Mean^(d)) Stimulant^(a) SPLC PPC SPLC PPC SPLC PPC HAV IR #1: None — — 2.0 ND 2.0 1.5 Mitogen (10 μg/mL) 0.9 4.5 1.7 ND ND ND HAV (11 μg/mL) 0.6 2.1 1.1 ND ND ND HAV (5.5 μg/mL) 0.8 3.0 1.3 ND ND ND HAV (2.8 μg/mL) 1.1 3.9 1.1 ND ND ND HAV (1.4 μg/mL) 0.6 4.1 1.2 ND ND ND HAV IR #2.: None — — 1.1 ND 0 0.3 Mitogen (10 μg/mL)^(c) 1.1 4.1 1.3 ND ND ND HAV (11 μg/mL) 1.0 0.8 1.2 ND ND ND HAV (5.5 μg/mL) 0.9 1.2 1.3 ND ND ND HAV (2.8 μg/mL) 0.7 0.6 1.0 ND ND ND HAV (1.4 μg/mL) 0.7 1.0 1.5 ND ND ND HAV IR #3.: None — — 1.5 ND 4.8 0 Mitogen (10 μg/mL)^(e) 2.1 0.5 1.5 ND ND ND HAV (11 μg/mL) 2.0 0.5 1.5 ND ND ND HAV (5.5 μg/mL) 2.3 0.7 1.3 ND ND ND HAV (28 μg/mL) 8.0 1.1 1.3 ND ND ND HAV (1.4 μg/mL) 2.2 1.3 1.3 ND ND ND HAV IR #4.: None — — 1.2 ND 1.0 2 Mitogen (10 μg/mL)^(e) 1.7 2.4 1.3 ND ND ND HAV (11 μg/mL) 2.4 1.0 1.3 ND ND ND HAV (5.5 μg/mL) 1.7 1.2 1.3 ND ND ND HAV (2.8 μg/mL) 8.0 1.1 1.3 ND ND ND HAV (1.4 μg/mL) 1.7 1.4 1.2 ND ND ND HAV IR #5.: None — — 1.0 ND 0 0 Mitogen (10 μg/mL)^(e) 1.8 0.7 1.1 ND ND ND HAV (11 μg/mL) 1.2 0.5 1.8 ND ND ND HAV (5.5 μg/mL) 1.1 0.8 1.5 ND ND ND HAV (2.8 μg/mL) 1.1 0.8 1.8 ND ND ND HAV (1.4 μg/mL) 0.9 0.8 1.3 ND ND ND HAV IR #6: None — — 1.3 ND 0 0 Mitogen (10 μg/mL) 0.6 0.7 1.0 ND ND ND HAV (11 μg/mL) 0.6 0.8 1.3 ND ND ND HAV (5.5 μg/mL) 1.0 0.7 1.4 ND ND ND HAV (2.8 μg/mL) 0.7 0.9 1.4 ND ND ND HAV (1.4 μg/mL) 0.7 0.7 1.2 ND ND ND HAV IR #7: None — — 2.7 ND 5.8 0.5 Mitogen (10 μg/mL) 2.0 0.8 2.6 ND ND ND HAV (11 μg/mL) 1.6 1.1 2.7 ND ND ND HAV (5.5 μg/mL) 2.3 0.9 2.9 ND ND ND HAV (2.8 μg/mL) 1.6 0.9 2.7 ND ND ND HAV (1.4 μg/mL) 1.0 1.1 2.6 ND ND ND HAV IR #8: None — — 2.4 ND 2.5 7.5 Mitogen (10 μg/mL) 1.2 1.8 2.8 ND ND ND HAV (11 μg/mL) 1.2 1.9 2.4 ND ND ND HAV (5.5 μg/mL) 1.5 1.7 2.7 ND ND ND HAV (2.8 μg/mL) 1.0 3.1 2.8 ND ND ND HAV (1.4 μg/mL) 1.3 2.8 2.7 ND ND ND HAV IR #9: None — — 2.8 ND 1.3 2.0 Mitogen (10 μg/mL) 0.8 3.3 2.8 ND ND ND HAV (11 μg/mL) 0.8 3.1 2.8 ND ND ND HAV (5.5 μg/mL) 0.8 2.7 2.9 ND ND ND HAV (2.8 μg/mL) 0.8 3.1 3.6 ND ND ND HAV (1.4 μg/mL) 0.5 2.8 3.0 ND ND ND HAV IR #10: None — — 2.9 ND 0.5 4.5 Mitogen (10 μg/mL) 1.3 1.7 2.6 ND ND ND HAV (11 μg/mL) 1.2 1.1 2.8 ND ND ND HAV (5.5 μg/mL) 1.4 1.8 2.5 ND ND ND HAV (2.8 μg/mL) 1.2 1.5 2.9 ND ND ND HAV (1.4 μg/mL) 1.3 1.5 2.9 ND ND ND HAV PO#1: None — — 3.6 ND 1.5 7.8 Mitogen (10 μg/mL) 1.2 2.9 2.9 ND ND ND HAV (11 μg/mL) 0.9 11.6 2.6 ND ND ND HAV (5.5 μg/mL) 1.1 1.1 2.6 ND ND ND HAV (2.8 μg/mL) 0.7 0.9 2.5 ND ND ND HAV (1.4 μg/mL) 1.2 1.4 3.1 ND ND ND HAV PO#2: None — — 2.8 ND 2.3 11.8 Mitogen (10 μg/mL) 1.5 4.1 3.3 ND ND ND HAV (11 μg/mL) 1.2 1.7 2.7 ND ND ND HAV (5.5 μg/mL) 1.8 1.9 3.2 ND ND ND HAV (2.8 μg/mL) 0.9 2.0 2.7 ND ND ND HAV (1.4 μg/mL) 1.1 2.2 3.0 ND ND ND T12-2 PO #1.: None — — 2.6 ND 5.0 6.0 Mitogen (10 μg/mL) 0.1 1.6 3.2 ND ND ND HAV (11 μg/mL) 2.4 1.5 3.2 ND ND ND HAV (5.5 μg/mL) 2.5 1.0 3.0 ND ND ND HAV (2.8 μg/mL) 1.5 1.3 3.1 ND ND ND HAV (1.4 μg/mL) 2.0 1.9 3.1 ND ND ND T12-2 PO #2.: None — — 2.8 ND 5.0 3.0 Mitogen (10 μg/mL) 1.1 0.9 0.7 ND ND ND HAV (11 μg/mL) 1.0 0.8 2.5 ND ND ND HAV (5.5 μg/mL) 1.3 0.8 3.1 ND ND ND HAV (2.8 μg/mL) 1.0 1.1 3.0 ND ND ND HAV (1.4 μg/mL) 2.1 1.2 3.2 ND ND ND T12-2 PO #3.: None — — 2.3 ND 4.5 1.0 Mitogen (10 μg/mL) 1.1 2.3 2.3 ND ND ND HAV (11 μg/mL) 1.1 1.2 2.3 ND ND ND HAV (5.5 μg/mL) 1.0 1.6 3.9 ND ND ND HAV (2.8 μg/mL) 0.6 1.0 7.2 ND ND ND HAV (1.4 μg/mL) 0.9 2.3 2.1 ND ND ND T12-3 PO #1: None — — 2.6 ND 2.0 0 Mitogen (10 μg/mL) 1.5 1.6 2.4 ND ND ND HAV (11 μg/mL) 0.9 1.8 2.6 ND ND ND HAV (5.5 μg/mL) 1.1 1.2 2.0 ND ND ND HAV (2.8 μg/mL) 0.5 1.0 1.9 ND ND ND HAV (1.4 μg/mL) 0.5 1.3 1.8 ND ND ND T12-3 PO #2. None — — 0.5 ND 4.3 2.8 Mitogen (10 μg/mL) 0.8 2.2 2.1 ND ND ND HAV (11 μg/mL) 2.2 2.1 1.6 ND ND ND HAV (5.5 μg/mL) 2.1 2.2 2.2 ND ND ND HAV (2.8 μg/mL) 0.5 2.7 2.3 ND ND ND HAV (1.4 μg/mL) 0.7 2.0 1.8 ND ND ND T12-3 PO #3: None — — 2.0 ND 2.0 0.8 Mitogen (10 μg/mL) 1.1 1.6 1.5 ND ND ND HAV (11 μg/mL) 1.6 1.6 2.1 ND ND ND HAV (5.5 μg/mL) 1.7 1.1 2.0 ND ND ND HAV (2.8 μg/mL) 1.2 1.2 2.1 ND ND ND HAV (1.4 μg/mL) 1.0 1.3 2.1 ND ND ND

[0100] TABLE 8 Results: Negative Controls (not boosted). LST EIA ELISPOT Immunization Stimulation Specific IgA No.IgA-AFC Protocol/ Index (SI)^(b) (S/N)^(c) (Mean^(d)) Stimulant^(a) SPLC PPC SPLC PPC SPLC PPC Saline PO: None — — 1.6 ND Mitogen (10 μg/mL) 1.1 2.4 2.6 ND ND ND HAV (11 μg/mL) 2.0 1.9 1.9 ND ND ND HAV (5.5 μg/mL) 1.1 1.5 2.2 ND ND ND HAV (2.8 μg/mL) 0.3 0.9 1.9 ND ND ND HAV (1.4 μg/mL) 0.9 1.8 1.0 ND ND ND Saline IR#1: None — — 2.0 ND Mitogen (10 μg/mL) 1.3 1.5 2.4 ND ND ND HAV (11 μg/mL) 1.4 1.0 2.3 ND ND ND HAV (5.5 μg/mL) 1.7 1.1 3.0 ND ND ND HAV (2.8 μg/mL) 1.1 1.1 3.6 ND ND ND HAV (1.4 μg/mL) 0.8 1.4 2.1 ND ND ND Saline IR #2: None — — 2.2 ND Mitogen (10 μg/mL) 0.9 1.0 2.7 ND ND ND HAV (11 μg/mL) 1.4 0.8 2.1 ND ND ND HAV (5.5 μg/mL) 1.2 1.1 2.1 ND ND ND HAV (2.8 μg/mL) 0.9 1.0 2.0 ND ND ND HAV (1.4 μg/mL) 1.1 1.1 1.8 ND ND ND

[0101] Summary and Conclusions:

[0102] These table results are summarized as follows. The hepatitis A viral (HAV) antigen (purchased from Microbix as a semi-purified preparation grown in FRhK4 monkey kidney cells) is immunogenic in mice when delivered by the intraperitoneal (IP) route emulsified with incomplete Freund's adjuvant (IFA); intrarectally (IR) by direct application or orally (PO) by feeding. HAV antigen given by IP, IR, or PO routes generates HAV-responsive (memory) lymphocytes in both the spleen cell (SPLC) and Peyer's patch cell (PPC) populations that can be detected by in vitro proliferation assays or after in vivo boosting with HAV. In mice immunized via the mucosal (IR or PO) routes the proliferative response appears to be more prevalent and more vigorous in the PPC population than in the SPLC population, indicating that mucosal routes of immunization are effective at eliciting local (intestinal) immune responses. HAV antigen given by the IP, IR or PO routes also gives rise to HAV-specific IgA forming cells in both the SPLC and PPC populations which can be detected by ELISPOT assays either directly (after immunization) or indirectly (after in vitro boosting in bulk culture—data not shown). However, cells forming IgA that was reactive with FRhK4 antigens as well as other assay constituents are also detectable by ELISPOT assays. Therefore, results have been expressed as the net number of positive (HAV-specific) spots after subtraction of this background reactivity. HAV-specific cells present in the SPLC and PPC populations can be induced to synthesize and secrete HAV-specific IgA detectable by direct enzyme immunoassay (EIA) after in vitro stimulation in bulk culture with pokeweed mitogen (PWM or HAV antigen at various concentrations. Two transgenic tomato vaccines given orally also appeared to be immunogenic in that they provoked immunologic memory lymphocytes in the spleen and Peyer's patches that were detectable by proliferation assays.

[0103] 3.2.2. Further Examination of the Mucosal Immune Response to HAVRIX™ and the Effect of Dose on the Response to Rectal Immunization:

[0104] To determine the effect of HAVRIX™ dose on the intestinal mucosal and systemic immune responses to HAV, and to determine if rectal immunization induces immunologic memory to HAV, the following experiment was performed. In this experiment the methodology to assess the mucosal immune response was further refined to increase the sensitivity of detection of the immune response. This experiment compared the immune responses in both systemic and mucosal compartments to intrarectal (IR) immunization and immunization via conventional intraperitoneal (IP) routes. Immune responses were evaluated in systemic (spleen cell=SPLC) and gut mucosal (Peyer's patch cell=PPC, lamina propria lymphocyte=LPL) cell populations by three methods that were modified from the procedures described above:

[0105] 1. HAV-specific ELISPOT assays measuring the production of IgG, IgM, and IgA class specific antibody forming cells (AFC).

[0106] 2. Class-specific EIAs which measure in HAV-specific IgG, IgM, and IgA antibodies in both serum and fecal extracts (coproantibodies).

[0107] 3. HAV-specific lymphocyte stimulation assays that measured the in vitro proliferative response of lymphocytes. This assay is considered a surrogate measurement of the immune responsiveness of CD4+ T-cell helper cells in lymphoid populations.

[0108] The measured responses of SPLC and in the sera were considered indicative of the systemic immune response while that of the PPCs and LPLs was considered representative, respectively, of the inductive and effector compartments of the intestinal mucosal immune system

[0109] Immunization of Mice and Collection of Tissues:

[0110] Four doses of HAVRIX™ 1440 were compared: 144, 72, 36, and 18 EU. BALB/c female mice (Harlan Laboratories, Rehovot, Israel), 4-6 weeks at the outset of the experiment, in groups of 5, received three doses of HAVRIX™ (either IP or IR) at weekly intervals after which, they were allowed to rest for a period of 1-3 months (Table 9). Control animals received sterile saline via the IP or IR routes according to the same schedule. Subsequently, three animals in each group (including the saline controls) were boosted with 72 EU of HAVRIX™ given by the IP route (regardless of the route used for the priming immunization). This strategy was designed to mimic the situation in which the vaccine would be delivered parenterally (by injection), in an immunization campaign during an outbreak situation to previously immunized individuals. Two animals in each group were not immunized further. In the saline control groups, the immune responses of the three animals immunized with 72 EU of HAVRIX™ IP, were considered to be representative of a primary immune response to this dose/route of HAVRIX™, while the responses measured in the remaining saline controls represented nonspecific background in the assays used. Blood and fecal specimens (for the preparation of serum and coproantibodies—see below) were collected prior to, and 1 week after each immunization. Two weeks after delivery of the last (booster) dose of HAVRIX™, all animals in each group were killed. Blood was removed for preparation of sera, and the spleen and small intestine (from the pylorus to ileocaecal junction) were removed for analysis of systemic (spleen cell=SPLC) and gut mucosal (PPC, LPL) lymphocyte responses. The measured response of SPLC and serum antibody determinations were considered indicative of the systemic immune response while those of the PPCs, LPLs, and coproantibody determinations were considered representative of the inductive (PPC) and effector (LPL, coproantibodies) arms of the mucosal immune system. In contrast to the earlier experiments, animals were kept under SPF conditions and provided with autoclaved standard rodent chow and water ad libitum. TABLE 9 Treatment Groups. Number of Mice Route Priming Dose (EU)^(a) Total Boosted^(b) Not Boosted IP 144 5 3 2 72 5 3 2 36 5 3 2 18 5 3 2 0 5 3 2 IR 144 5 3 2 72 5 3 2 36 5 3 2 18 5 3 2 0 5 3 2

[0111] Serum and Coproantibody Preparation:

[0112] Mice were bled via the tail vein and serum was prepared and stored at −20° C. until testing. While the mice were being bled, fecal pellets were collected and coproantibodies were extracted as soon as possible according to the procedure described by DeVos, et al. DeVos T, Dick T A. A rapid method to determine the isotope and specificity of coproantibodies in mice infected with Trichinella or fed cholera toxin. J Immunol Meth 1991;141 285-288.).

[0113] Cell Preparations:

[0114] SPLC, PPC and LPL suspensions were prepared from individual mice, as follows. SPLC: Spleens were removed and placed into 60 mm petri dishes with 3-5 mL of sterile RPMI-1640 medium (GIBCO/BRL, Biological Industries, Beit Haemek, Israel) containing antibiotics penicillin, 100 U/mL and streptomycin, 100 μg/mL, GIBCO/BRL), placed on ice, and transported to the laboratory for further processing. The organs were washed once in sterile RPMI or Dulbecco's phosphate buffered saline (PBS), and then teased apart in 5 mL of RPMI with antibiotics. The suspended SPLC were collected by centrifugation, washed twice through RPMI with antibiotics, then resuspended in 10 mL of the same medium containing 2% heat-inactivated (56 C, 30 min) FBS (Biological Industries). Viable lymphoid cells were enumerated by Trypan Blue exclusion using a Neubauer hemocytometer. For ELISPOT and lymphocyte proliferation assays, SPLC were adjusted to 1×10⁶ cells/mL in RPMI medium containing antibiotics and 10% FBLS. PPC: Peyer's patches (approx. 4-8 per animal) were removed from the serosal surface of the gut into sterile 60-mm petri dishes containing 5 mL of sterile RPMI-1640 medium with antibiotics. The organs were washed once in sterile RPMI or sterile PBS then teased apart in 5 mL of the same medium containing 1.5 mg/mL of Dispase (Sigma-Aldrich, Milwaukee, Wis.). The cell suspension was transferred into a conical-bottomed 10-15 mL tube and incubated with shaking for 45-60 min at 37° C. to dissociate lymphoid cells from the rest of the tissue. PPCs were collected by centrifugation and washed twice through RPMI with antibiotics. Numbers of viable lymphoid cells were determined by Trypan Blue exclusion. As PPC numbers were limited, the final pellet was resuspended in 2.0-2.5 mL of RPMI with antibiotics and 10% FBS for subsequent use in ELISPOT and lymphoproliferation assays. Cell densities (cells/mL) for each PPC preparation were recorded for later normalization of assay results to the estimated response had the initial cell density been 1×10⁶ cells/mL, to allow for data comparisons.

[0115] LPLs: A modification of the method of Jackson, et al. (Jackson R J, Fujihashi I, Xu-Amano H, Kiyono H, Elson C O, McGhee J R. Optimizing Oral Vaccines: Induction of Systemic and Mucosal B-cell and Antibody Responses to Tetanus Toxoid by Use of Cholera Toxin as an Adjuvant. Infect Immun 1993;61(10):4272-79) was used to extract LPLs from the small intestine wall. The entire small intestine was opened longitudinally and washed thoroughly with RPMI-1640 medium or sterile PBS, then cut into segments 2-3 cm in length. To remove intraepithelial lymphocytes (IELs) and other lymphoid cells (principally, CD9+ T cells) located in intestinal mucosal crypts, tissues from individual animals were placed in 10 mL of in RPMI with 2% FBS in 50-mL centrifuge tubes and gently agitated on a mechanical shaker for 30 min at 37° C. At the end of this period, the tubes were shaken vigorously for 15 sec. Tissues were removed with the aid of a fine mesh strainer, then placed in fresh medium and shaken again for 15 sec, strained through the mesh, and transferred into fresh 50-mL tubes for further extraction in 10 mL of RPMI with antibiotics containing 1.5 mg/mL of Dispase. Enzymatic digests were carried out in three cycles 30 min at 37° C. on a mechanical shaker. At the end of each incubation period, the LPLs present in the supernatant were collected by centrifugation, resuspended in RPMI with antibiotics and 2% FBS and placed on ice until the third digestion was complete. The LPLs were pooled and collected by centrifugation. Numbers of viable lymphoid cells were determined by Trypan Blue exclusion hemocytometer counts, cells were resuspended in 2-2.5 mL of RPMI with antibiotics and 10% FBS and used at their available densities for ELISPOT and lymphocyte proliferation assays. Individual densities were recorded and the data were normalized to the estimated response for an initial cell density of 1×10⁶ cells/mL for subsequent data comparisons.

[0116] Immunoassays:

[0117] HAV-specific systemic and mucosal immune responses were evaluated by three methods i) ELISPOT assays to determine the frequencies of IgG, IgM, and IgA class antibody forming cells (AFC); ii) enzyme immunoassays (EIAs) which measured antigen-specific IgG, IgM, and IgG antibodies in both serum and fecal extracts (coproantibodies); iii) lymphocyte stimulation assays that measured the in vitro dose-related proliferative response of lymphocytes to HAV antigen.

[0118] Antigen: The HAV antigen used for these immunoassays was highly purified alum-free (to avoid possible interference of this adjuvant with the antigenicity of the virus in immunoassays) formalin-fixed HAV purchased from a vaccine manufacturer (Aventis-Pasteur, Lyon, France). The concentration of HAV in the undiluted preparation was 1064 EU/mL or 170 μg of protein/mL.

[0119] ELISPOT Assays:

[0120] Numbers of HAV-specific IgG, IgM, or IgA AFC in SPLC, PPC, and LPL cell populations were determined by the method of Jackson, et al. (Jackson R J, Fujihashi I, Xu-Amano H, Kiyono H, Elson C O, McGhee J R. Optimizing Oral Vaccines: Induction of Systemic and Mucosal B-cell and Antibody Responses to Tetanus Toxoid by Use of Cholera Toxin as an Adjuvant. Infect Immun 1993;61(10):4272-79) Briefly, wells of sterile 96-well nitrocellulose-bottomed microplates (MAHAN S4510, Multiscreen HA plate Millipore, Bedford, Mass.) were coated for 24 h at 4° C. with 100 μL of HAV antigen diluted to a concentration of 5 μg/mL in sterile bicarbonate-carbonate coating buffer (pH 9.6). The antigen was discarded and the wells were washed three times in sterile Dulbecco's phosphate buffered saline (PBS, pH 7.4) then blocked for a minimum of 1 h at room temperature with sterile 1% bovine serum albumin (BSA) in PBS. Just before adding the cells to be tested, the blocking solution was discarded with no further washing of the microplate wells. All cell populations were added such that the well volume was 100 μL regardless of the cell densities of the SPLC, PPC, or LPL populations. The numbers of SPLC added per well were 10,000 for all preparations, while the numbers of PPC and LPL varied according to the yield for individual preparations. Regardless of the cell numbers, data were normalized to an estimate of the response that would be obtained if 100,000 cells were added to each well for each PPC or LPL preparation. Control wells containing reagent only were included on each plate to distinguish antigen-specific from nonspecific spots. Each cell preparation was tested in duplicate. After adding the cells, the plates were transferred carefully to a 37 C, 5% CO₂ incubator and left overnight. The following day, the cells were discarded and the wells were washed with three changes of sterile PBS, then three times with PBS containing 0.05% Tween 20 (PBST). To detect HAV-specific antibodies of the IgG, IgM, and IgA classes, horseradish peroxidase (HSP)—conjugated goat anti-mouse class-specific antibodies (Southern Biotechnology Associates [SBA], Birmingham, Ala.) diluted to 1:1000, each, in PBST containing 5% normal goat serum (NGS) were added (100 μL/well) and is incubated for 18 hours at 4 C. These antibodies were discarded and the wells were washed 4 times with PBST. To visualize ELISPOTs indicative of positions of HAV-specific AFC, 3-aminoethyl-9-carbazole substrate (AEC, Sigma-Aldrich) diluted in 0.1 M acetate buffer containing 0.015% H₂O₂(100 μL/well) was added. The plates were covered with aluminium foil and incubated at room temperature for 20 min. The enzymatic reaction was stopped, by washing the wells 4 times with distilled water. ELISPOTs were enumerated using a top-illuminated dissecting microscope. Characteristic ELISPOTs were not observed in control wells. Results were expressed as the averaged value of duplicate wells normalized to the estimated number of spots per 10⁶ cells and corrected for background (i.e., the averaged number of ELISPOTs observed in non-immunized saline controls were subtracted to provide a net value).

[0121] EIAs: HAV-specific antibodies of the IgG, IgM, and IgA classes were determined by a direct EIA. Briefly, wells of 96-well, flat-bottomed polystyrene microplates (Nunc ImmunoPolysorp, Danyel Biotech, Rehovot, Israel) were coated with HAV antigen diluted to a concentration of 10 μg/mL in bicarbonate-carbonate buffer, pH 9.6 (100 μL/well) overnight at 4 C. The antigen was discarded and the plates were blocked for 1 h with 3% nonfat milk in PBS (pH 7.4) (200 μL/well). The blocking solution was discarded and the wells were washed three times with PBST. For serum antibody determinations serial 2-fold dilutions (in blocking buffer) ranging from 1:50 to 1:6400 were prepared directly in the wells such that the final well volume was 100 μL. A preimmunization BALB/c mouse serum pool diluted the same, was included as a control in addition to a row of wells that received dilution buffer only. Fecal extracts for coproantibody determinations were added undiluted (100 μL/well). Negative controls for coproantibody determinations included Hank's Balanced Salt solution (HBSS), the extraction buffer used in preparing coproantibodies; fecal extracts from non-immunized saline control mice; and extracts of feces collected from 30 of the mice before immunization. All determinations on each plate were single. However, replicate assays were performed.

[0122] The plates were incubated for 2 h at room temperature or overnight at 4° C. Following this, the weft contents were discarded and wells were washed 4 times with PBST. To detect class-specific antibodies, BRP-goat anti-mouse IgG, IgM, or IgA (from SBA), diluted 1:1000 in 5% NGS in PBST were added to each well (100 μL/well) and the plates were incubated for 2 h at room temperature. The well contents were discarded, and the plates were washed 4 times with PBST. HRP substrate (ortho-phenylenediamine in 0.1 citrate-phosphate buffer (pH 5) containing 0.015% H₂O₂ (100 μL/well) was added and the plates were incubated at room temperature for 60-90 min. A₄₅₀ was determined for each well using a Spectra Rainbow microplate reader. Endpoint titers for serum determinations were expressed as the last serum dilution showing an absorbance value equal to or greater than the mean plus two standard deviations of the averaged absorbance values calculated for both sets of control wells. Results for coproantibody determinations were expressed qualitatively as positive or negative based on a signal-to-noise (S/N) calculated from the absorbance measured in wells containing fecal extracts divided by the averaged absorbance measured in all the negative control wells S/N ratios>2 were taken to be positive for IgG and IgM determinations, while S/N ratios>3 were taken to be positive for IgA determinations.

[0123] Lymphocyte Proliferation Assays:

[0124] The in vitro response of SPLC, PPC, and LPLs to stimulation with formalin-inactivated HAV was determined using standard lymphocyte proliferation assays performed in U-bottomed 96-well microculture plates. HAV antigen was diluted to give final well concentrations of 20, 10, 5, and 2.5 EU/mL. Positive control wells received PHA (Sigma-Aldrich) adjusted to a final concentration of 10 μg/mL. Negative control wells received medium only. Cells were added in a fixed volume of 100 μL regardless of the density in the original preparation. Thus, SPLC were added at an initial density of 100,000 cells per well while PPC and LPL initial densities varied according to individual yields. Results were normalized to the response estimated if the initial well density had been 100,000 cells/well. Well volumes were brought up to 0.25 mL with RPMI containing antibiotics and 10% FCS (as described above) and the cells were incubated at 37° C. in 5% CO₂ in air for 7 days. During the last 18 hours of culture, the cells were pulsed with 1 μCi/well of ³H-thymidine (Amersham, Piscataway, N.J.). The cells were harvested by hypotonic lysis and the DNA collected onto glass fiber filters for counting (Wallac Microbeta Harvestor and Counter, Turku, Finland). Results were expressed as the stimulation index (SI) calculated from the averaged cpm per well containing stimulant divided by the averaged cpm of negative control (medium only) wells and corrected for averaged background values observed in non-immunized saline controls.

[0125] Statistical Methods:

[0126] Frequencies of AFCs and serum endpoint titers (taken as absolute values) were compared using 1-tailed, 2-sample T-tests for equal or unequal variance. F-tests were used to determine equal or unequal variance.

[0127] Results:

[0128] Determination of HAV-Specific Antibodies by EIA and ELISPOT Assays:

[0129] HAV-specific antibodies of IgG, IgM, and IgA classes were measured in both sera and fecal extracts (coproantibodies) by EIA to evaluate the specific antibody response in the systemic and intestinal mucosal immune compartments, respectively. Similarly, systemic (SPLC) and mucosal (PPC, LPL) AFC were enumerated by ELISPOT assays. Results comparing the response of intraperitoneally (IP) and intrarectally (IR) immunized animals are summarized for each HAVRIX™ immunizing dose, successively, in Tables 10-14. ELISPOT values shown in the Tables have been corrected for background levels of activity observed in saline control mice that received saline only, throughout the course of the experiment. Coproantibodies are expressed qualitatively as either present (+) or absent (−).

[0130] Response to 144 EU of HAVRIX™ (Table 10):

[0131] While the mean numbers of IgG AFCs found in SPLC of mice immunized via the IP route appeared to be higher relative to those observed in IR-primed animals this difference was not statistically significant (p=0.132). HAV-specific IgG AFC were not observed in PPC and LPL populations in either group. Large numbers of IgM AFC were found in the spleens of both groups. However, no significant group differences were observed. IgM AFC were also found in PPC and LPL populations of some rectally immunized mice. IgA AFCs were not observed in SPLC of any of the animals. Nor were they found in the PPCs of IP-immunized mice. However, they were found in abundance in both the PPC and LPL populations of rectally immunized mice and at a significantly higher (p=0.043) frequency in the PPC population (LPLs, not compared). Correspondent with observed higher IgG AFC frequencies, titers of HAV-specific IgG while appearing higher in IP-immunized mice, were not found to be significantly different from those of the IR group (p=0.094). Titers of specific IgM antibodies were higher in the sera of IR-immunized mice (p=0.009) in keeping with the consistently large numbers of IgM AFC seen in the spleens of this group. While titers of HAV-specific IgA serum antibodies also appeared higher in the IP-immunized mice, they were not significantly different from the IR group (p=0.091) IgG and IgA-class HAV-specific coproantibodies were detected in fecal extracts from 1/5, and 2/5 rectally immunized mice, respectively, but not in any animals from the IP group. IgM class coproantibodies were not observed in any of the mice. The additional dose of 72 EU of HAVRIX™ (given IP to Animals 1-3 in each group) two weeks before killing the mice did not appear to enhance the frequencies of AFC, in comparison to those found in Animals 4 and 5 in each group who received only three doses of HAVRIX™. TABLE 10 Comparative levels of HAV-specific IgG, IgM, and IgA class antibodies determined in mice primed intraperitoneally (IP) or intrarectally (IR) with 144 EU of HAVRIX ™. Animal IP Route IR Route Number AFC/10⁶ cells Titer⁻¹ Copro- AFC/10⁶ cells Titer⁻¹ Copro- (Treatment) SPLC PPC LPLs (serum) Ab SPLC PPC LPLs (serum) Ab IgG Antibodies: 1 (B) 35 0 ND  800 − 0 0 0  <50 − 2 (B) 5 0 ND ≧6400  − 0 0 0 ≧100 + 3 (B) 5 0 ND ≧800 − 10 0 0  <50 − 4 (NB) 5 0 ND ≧200 − 0 0 0  <50 + 5 (NB) 0 0 ND ≧400 − 0 0 0  ≧50 − IgM Antibodies: 1 (B) 290 0 ND  ≧50 − 57.5 0 0 ≧200 − 2 (B) 0 0 ND ≧100 − 0 0 6 ≧200 − 3 (B) 15 0 ND ≧100 − 62.5 8.5 0 ≧200 − 4 (NB) 15 0 ND  50 − 22.5 0 0 ≧100 − 5 (NB) 0 0 ND  50 − 67.5 0 0 ≧100 − IgA Antibodies: 1 (B) 0 0 ND  <50 − 0 0 0  <50 − 2 (B) 0 0 ND 1600 − 0 28 0  <50 − 3 (B) 0 0 ND ≧800 − 0 20 0  <50 − 4 (NB) 0 0 ND ≧1600  − 0 0 14  <50 ND 5 (NB) 0 0 ND  800 − 0 38 146 ≧1600  +

[0132] Response to 72 EU of HAVRIX™ (Table 11):

[0133] Again, HAV-specific IgG and IgM AFC frequencies in SPLC were higher in IP-immunized animals than in rectally immunized mice. This was of borderline statistical significance (IgG: p=0.053; IgM: p=0.057). Fewer IgA AFC were observed in SPLC of both treatment groups (no difference). In PPC populations, one animal in the IP-immunized group had a large number of IgM AFC (no significant group differences overall). Too few LPLs were isolated from the IP-primed animals, hence, group comparisons for AFC were not possible. However, IgA AFC were observed in PPC and LPL preparations from two rectally immunized mice. Serum titers of HAV-specific IgG (p=0.014) and IgA (p=0.038) were higher in mice immunized via the IP route compared to the IR group. No group differences were observed for specific IgM levels. Again, IgG, IgM, and IgA class HAV-specific coproantibodies were observed only in rectally immunized mice. The extra dose of 72 EU of HAVRIX™ (given IP to Animals 1-3 in each group) did not appear to enhance the frequencies of AFC or antibody levels in either IP- or IR-immunized mice. TABLE 11 Comparative levels of HAV-specific IgG, IgM, and IgA class antibodies determined in mice primed intraperitoneally (IP) or intrarectally (IR) with 72 EU of HAVRIX ™. Animal IP Route IR Route Number AFC/10⁶ cells Titer⁻¹ Copro- AFC/10⁶ cells Titer⁻¹ Copro- (Treatment) SPLC PPC LPLs (serum) Ab SPLC PPC LPLs (serum) Ab IgG Antibodies: 1 (B) 8 0 ND  800 − 0 0 0  100 − 2 (B) 35 0 ND ≧3200  − 15 0 0 ≧100 − 3 (B) 5 0 ND 6400 − 5 0 16  800 + 4 (NB) 18 0 ND ≧1600  − 5 9 0  <50 − 5 (NB) 20 0 ND 3200 − 5 0 0  <50 − IgM Antibodies: 1 (B) 70 1.5 ND ≧100 − 0 0 0 ≧100 − 2 (B) 170 4.5 ND ≧100 − 47.5 0 0  400 − 3 (B) 5 148.5 ND ≧100 − 67.5 0 0 ≧100 − 4 (NB) 100 0 ND  100 − 42.5 6.5 0  200 + 5 (NB) 105 2.5 ND  50 − 17.5 0 0  <50 − IgA Antibodies: 1 (B) 2.5 0 ND ≧200 − 0 18 0  <50 − 2 (B) 0 0 ND ≧400 − 0 28 0  <50 − 3 (B) 0 0 ND ≧200 − 0 0 59 ≧400 + 4 (NB) 0 0 ND 1600 − 5 0 53  <50 − 5 (NB) 0 0 ND  800 − 5 11 7  <50 −

[0134] Response to 36 EU of HAVRIX™ (Table 12):

[0135] The frequencies of specific IgG AFC in spleens of IP-primed animals were higher (p=0.048) than that of their IR counterparts. Specific IgG AFC were also observed in PPC populations of one IP-primed mouse and two IP-immunized mice and while higher in the latter group, were not significantly different. Frequencies of specific IgM AFC were high in both groups (not significantly different). Specific IgA AFCs were also observed in all cell populations in both groups (no significant differences). However, 3 of the PPC, and 4 of the LPL preparations obtained from rectally immunized mice had strikingly high frequencies of specific IgA AFC. Serum titers of specific IgG antibodies were also found to be significantly higher (p=0.035) in the IP group compared to rectally immunized mice. Titers of specific IgM antibodies were also higher (p=0.033) in the IP group. In contrast, serum titers of HAV-specific IgA antibodies were significantly higher (p=0.032) in rectally immunized mice. IgA coproantibodies were found in fecal extracts of 3/5 rectally immunized mice but only one in the IP group. TABLE 12 Comparative levels of HAV-specific IgG, IgM, and IgA class antibodies determined in mice primed intraperitoneally (IP) or intrarectally (IR) with 36 EU of HAVRIX ™. Animal IP Route IR Route Number AFC/10⁶ cells Titer⁻¹ Copro- AFC/10⁶ cells Titer⁻¹ Copro- (Treatment) SPLC PPC LPLs (serum) Ab SPLC PPC LPLs (serum) Ab IgG Antibodies: 1 (B) 0 0 ND ≧400 − 0 0 0 ≧100 − 2 (B) 10 0 ND 3200 − 5 0 0  <50 − 3 (B) 0 6 ND ≧6400  − 0 24 0  <50 − 4 (NB) 10 0 ND 1600 − 0 0 0  100 − 5 (NB) 10 0 ND ≧400 − 0 10 0  <50 − IgM Antibodies: 1 (B) 0 0 ND  200 − 32.5 0 0  50 − 2 (B) 5 0 ND  400 − 32.5 0 0  ≧50 − 3 (B) 75 0 ND 1600 − 32.5 12.5 25 ≧100 + 4 (NB) 30 5.5 ND ≧400 − 0 0 12  50 − 5 (NB) 60 13.5 ND  400 − 27.5 0 39  50 − IgA Antibodies: 1 (B) 0 0 ND  <50 + 0 10 124 ≧400 + 2 (B) 0 0 ND  <50 − 5 0 506 ≧400 + 3 (B) 2.5 0 ND  <50 − 0 60 6 ≧400 − 4 (NB) 7.5 80.5 ND  200 − 0 0 53  <50 − 5 (NB) 0 0 ND  <50 − 0 31 144 ≧100 +

[0136] Response to 18 EU of HAVRIX™ (Table 13):

[0137] IgG AFCs were not found in SPLC of any mice immunized with 18 EU of HAVRIX™. Only a few IgG AFCs were observed in mucosal cell populations. IgM AFC frequencies appeared higher (but were not significantly so) in PPC populations from IP-immunized mice, whereas IgM AFC frequencies were significantly higher (p=0.003) in the spleens of the IR group. Only two animals in the i.p.-primed group demonstrated HAV-specific IgA AFC, and only in their LPLs, However, compared to the IP group, significantly higher (p=0.021) frequencies of IgA AFC in LPL populations were found in 4/5 rectally immunized mice, consistent with was observed in mice immunized with 36 EU of HAVRIX™ via the IR route. No significant group differences were observed in serum titers of HAV-specific IgG (p=0.282), IgM (p=0.116), or IgA (p=0.075) class antibodies. Coproantibodies of the IgA class were detected in fecal extracts from two rectally immunized mice. IgM coproantibodies were found in the fecal sample of one rectally immunized animal while IgG coproantibodies were detected in the feces of one of the IP group. TABLE 13 Comparative levels of HAV-specific IgG, IgM, and IgA class antibodies determined in mice primed intraperitoneally (IP) or intrarectally (IR) with 18 EU of HAVRIX ™. Animal IP Route IR Route Number AFC/10⁶ cells Titer⁻¹ Copro- AFC/10⁶ cells Titer⁻¹ Copro- (Treatment) SPLC PPC LPLs (serum) Ab SPLC PPC LPLs (serum) Ab IgG Antibodies: 1 (B) 0 0 ND ≧400 − 0 0 0  400 − 2 (B) 0 0 0 ≧1600  − 0 0 0 ≧1600  − 3 (B) 0 0 0 1600 − 0 0 0 ≧3200  − 4 (NB) 0 0 0 ≧800 + 0 5 11 ≧200 − 5 (NB) 0 0 13  ≧800 − 0 0 0 1600 − IgM Antibodies: 1 (B) 0 0 0  100 − 32.5 0 0  50 − 2 (B) 5 11.5 0  50 − 92.5 0.5 0  400 + 3 (B) 10 6.5 0  100 − 42.5 0 0  100 − 4 (NB) 0 20.5 23   <50 − 82.5 8.5 21  ≧50 − 5 (NB) 20 104.5 0  <50 − 67.5 0 11  100 − IgA Antibodies: 1 (B) 0 0 5 ≧800 − 0 16 68  <50 − 2 (B) 0 0 0 ≧1600  − 0 0 174  400 + 3 (B) 0 0 0  <50 − 0 0 0 ≧400 − 4 (NB) 0 0 0 6400 − 0 0 44  <50 − 5 (NB) 0 0 26  ≧3200  − 0 0 107 ≧800 +

[0138] Explanatory Notes (Tables 10-13):

[0139] SPLC: spleen cells; PPC: Peyer's Patch Cells; LPLs: lamina propria lymphocytes. Values shown are estimated frequencies of antibody-forming cells (AFC) per million cells in the three different cell population as determined by ELISPOT assays at the time of sacrificing the animals (see Methods).

[0140] Treatment: B=animals received 72 EU of HAVRIX™, i.p. two weeks before sacrificing. NB=animals were not immunized further after the initial 3-dose immunization schedule with HAVRIX™.

[0141] Serum titers: represent the reciprocal value of the endpoint titer measured in EIAs with serum dilutions ranging from 1:50 to 1:6400 (see Methods). Reciprocal titers<50 represent a negative value in the assay.

[0142] Coproantibodies: HAV-specific antibodies secreted into, and extracted from, feces (see Methods). Fecal samples were not obtained from all animals. Antibody levels which were measured by EIA are expressed qualitatively based on a calculated signal:noise (S/N) ratio. S/N>2 was considered representative of a positive response in IgG and IgM determinations; a S/N ratio of >3 was considered positive in assays for class-specific IgA antibodies (see Methods) Values shown represent those measured in fecal samples 1 week after boosting (Animals 1-3) and in nonboosted control animals at the same interval. Specific antibodies were not measurable in samples collected at earlier intervals.

[0143] ND: not determined.

[0144] FIGS. 12-14 summarize these observations of the antibody response to various immunizing doses of HAVRIX™ showing that at the highest dose (144 EU) IgG-AFC frequencies in SPLC populations were observed to be higher overall in mice immunized parenterally (via the IP route) in comparison to rectally immunized mice (FIG. 12), although these differences were not statistically significant. However, a lower immunizing doses of HAVRIX™ (36 and 18 EU), a strong reverse trend in frequencies of IgM- and IgA-AFC was observed, particularly in the LPL populations (FIGS. 13 and 14, respectively) which were observed to be significantly higher in rectally immunized mice in comparison to those who were immunized intraperitoneally.

[0145] Cellular Recognition of HAV Antigens in Immunized Mice as Determined by Lymphocyte Proliferation Assays

[0146] The in vitro dose-related responses of lymphocytes present in SPLC, PPC, and LPL populations of mice immunized with the varying doses of HAVRIX™ by the IP and IR routes were compared using conventional proliferation assays. As cell numbers in the PPC and LPL populations were limited (on the average, permitting an initial well density of 50,000 cells) incubations were carried out for 7 days. SPLC that were not limited in number, were plated at an initial density of 100,000 cells per well and results from wells containing PPC and LPLs were normalized to estimate the response at the same initial density. Insufficient cell numbers in some of the LPL populations and technical limitations reduced the numbers of comparisons using stimulation indices (SI) as the parameter. Individual data, summarized by immunizing dose and route, are shown in Table 14. In common practice, SI's>2.0 are considered to be indicative of a positive response, although some interpretations require higher cutoffs. All SI's shown in Table 14 have been corrected for assay background (i.e., SI's determined for the same cell populations obtained from non-immunized saline control mice have been subtracted) and therefore, this interpretation is considered valid here. In order to present the data comparatively, only the maximum SI's attained in the dose response curve for each cell preparation and for each animal, are shown in Table 14 and are summarized for all immunizing doses of HAVRIX™ in FIGS. 15-17. At higher immunizing doses of HAVRIX™ (144 and 72 EU) SI's were observed to be low or negative (with a few exceptions), in all cell populations. There were no discernible differences in the response related to the immunizing dose or route of vaccine delivery. At lower immunizing doses (36 and 18 EU) the SI's measured in all cell populations were observed to be 3—>100-fold higher in animals who were immunized via the IR route than those receiving HAVRIX™ via the IP route. The strength of the in vitro response of SPLC, PPC, and LPLs was also dose-related to the amount of stimulating HAV added to the cultures (data not shown). The highest SI's were observed in animals immunized with three intrarectal doses of 36 EU of HAVRIX™ (Table 14). While, differences in SI's measured in animals primed with 18 EU of HAVRIX™ via the IP and IR routes were not as striking as those observed with an immunizing dose of 36 EU, they were still at least 10-fold higher in the IR group, than in the IP groups. There were no discernible differences between the response of animals who received only three immunizations and those who received an additional booster dose of HAVRIX™. TABLE 14 Comparative in vitro response to HAV antigens of lymphocytes from mice immunized intraperitoneally (IP) or intrarectally (IR) with varying doses of HAVRIX ™ vaccine. HAVRIX ™ Maximal Stimulation Index (SI) Observed^(a) Dose/Cell IP Route IR Route Population 1 2 3 4 5 1 2 3 4 5 HAVRIX ™ 144 EU: SPLC ND ND 1.9 ND 23.6  10 0 0 0.3 ND PPC 0 ND 2.4 0 0.5 4.7 ND 2.7 0 ND LPLs ND ND ND ND ND ND ND 1.8 0 0 HAVRIX ™ 72 EU: SPLC ND ND 5.6 ND 0.5 0 ND 0 0 ND PPC 14.7 0.1 0 0 0.5 2.4 ND 0 0 ND LPLs ND ND ND ND ND 0 ND 26.5 ND 0 HAVRIX ™ 36 EU: SPLC 0.9 1.6 0.9 1.0 1.0 29.7 ND 12.5 166.7 16.8 PPC 4.6 1.5 2.9 2.8 7.4 12.3 ND 1.0 133.9 182.9 LPLs ND ND 2.4 ND ND ND 23.0 80.8 14.2 53.1 HAVRIX ™ 18 EU: SPLC 11.7 1.4 3.6 6.3 4.0 60.0 ND 55.1 17.6 1.6 PPC 6.4 0.8 1.9 9.8 3.4 23.0 ND 0 22.8 11.7 LPLs 5.4 1.6 2.7 3.8 2.5 2.8 62.2 44.5 ND 3.1

[0147] Footnotes (Table 14):

[0148]^(a) Value shown is the maximum stimulation index (SI) observed in the dose response to in vitro stimulation of lymphoid cells (see Methods) in the cell populations indicated in the first column of the table, compared for each treatment group (IP vs. IR) for each animal (1-5) in the treatment groups.

[0149] ND: not determined due to insufficient cell numbers or other technical limitations.

[0150] Summary and Conclusions:

[0151] The results of study indicate that immunization of mice with low doses of HAVRIX™ delivered via the intrarectal (IR) route appears to be highly effective in inducing HAV-specific IgA AFC (in mucosal lymphocytes) and IgM AFC (in splenocytes), as well as HAV-specific antibodies of these classes in the serum and intestinal mucosal secretions. This is desirable for immune protection as both pathogen blocking and neutralize antibodies are likely to be of these classes. Similarly, oral administration of the polio vaccine is known to elicit antibodies of both the IgG and IgA classes (see, for example; Herremans T M, Reimerink J H, Buisman A M, Kimman T G, Koopmans M P. Induction of mucosal immunity by inactivated poliovirus vaccine is dependent on previous mucosal contact with live virus. J Immunol. 1999 15;162:5011-8). For viruses that enter through the gut epithelium, mucosal IgA antibodies that are produced in the intestinal mucosa and secreted to the gut lumen, play an essential role in neutralizing the virus at the mucosal interface. Intrarectal immunization with HAV vaccine also induced strong cellular (as evidenced by lymphocyte proliferation) immune responses in both systemic (SPLC) and mucosal (PPC, LPL) lymphocyte populations that were superior to those responses observed in parenterally (i.p.) immunized animals. This suggests that rectal immunization has excellent potential for induction of T-cell help (either for production of specific antibodies or for induction of a cytotoxic T-lymphocyte response). Both antibody and cellular responses to HAVRIX™ administered by the rectal route were optimal at lower doses of immunizing antigen providing some encouragement that low doses of antigen (at least, by the IR route) should be effective rather than tolerizing. Also, these superior responses were manifest in both local (gut mucosa) and systemic (spleen) compartments of the immune system, and hence, represent a desirable means of inducing protection vs. HAV, a pathogen which enters the body via the gut mucosa and spreads to the liver.

[0152] Parenteral administration of vaccines via intradermal (ID) or intramuscular (IM) routes usually gives rise to only a systemic immune response consisting of IgM followed by IgG class antibodies as evidenced from results of IM administration of killed (Salk) polio vaccines in contrast to the oral (Sabin) polio vaccine that also induces IgA-class antibodies. Currently used injectable HAV vaccines that are also routinely delivered by IM injection are thought to produce only IgG class antibodies. Successful immunization against pathogens such as poliovirus and HAV that enter via the gut epithelium clearly requires local production of neutralizing antibodies of the IgA class at the portal of entry. Mucosal immunization via the oral or intrarectal, or intranasal routes induces both local (mucosal) and systemic immune responses consisting of both IgG and IgA antibodies. Thus, in the case of pathogens entering via mucosal (respiratory, gut, genitourinary) routes the latter type of immune response is clearly more beneficial to the host.

[0153] The method of the present invention, in which the HAV vaccine is administered to the body through the rectal mucosa, could have a major advantage over the current vaccination program, by not only by inducing antibody and cellular responses which block pathogen entry through the gastrointestinal mucosal but also by inducing systemic immune responses that would limit viral survival in the blood stream and/or its replication in liver tissue. In addition, the method of the present invention is able to induce the generation of a successful immune response against HAV without requiring needles or other invasive methods. The present invention thus enables the rectal administration of an HAV vaccine such as HAVRIX™ for the generation of a protective immune response against HAV infection.

[0154] Without wishing to limit the present invention, a suitable dosage of the HAV vaccine is preferably in the range of from about 0.75 to about 7500 EU of the antigen, for each administration more preferably approximately from about 50 EU to about 125 EU, and most preferably approximately 75 EU, applied to the rectum in a suppository cream, liquid, tablet, or any other solid, semi-solid or liquid dosage form which is suitable for the administration of HAV antigen to the rectal mucosa. Such a dosage form is well known in the art, and could easily be selected by one of ordinary skill in the art. Optionally the dosage form would contain an adjuvant, although alternatively, no adjuvant would be used.

[0155] The method of the present invention is also optionally and preferably suitable for the administration of at least one viral encapsidated gene as described above through the gastrointestinal mucosa of the subject, and particularly through the rectal mucosa. In this optional but preferred method, the viral encapsidated gene or genes is administered to the gastrointestinal mucosa of the subject, in a substantially similar manner as for the previously described HAV vaccine. Thus, the present invention also provides a method for administering one or more viral encapsidated gene or genes to the subject through the gastrointestinal, and particularly the rectal, mucosa.

[0156] Section 4:Future Implementations

[0157] The previously described transgenic plant vaccine contains non-infectious viral particles. These viral particles may also optionally be used to package other viral or host genes that have been engineered into HAV (or other viral) genomes that could subsequently, be engineered into tomato and or other plants using the Agrobacterium transformation system as demonstrated in this model. In this case, eating such transgenic tomatoes or other plant material, or otherwise applying the transgenes orally and/or rectally, would permit the transgenes to be specifically targeted to the liver by using HAV virus. Similarly, other viruses with specific trophism for bone marrow or nervous system could be employed in plant engineering systems to deliver specific genes to these tissues.

[0158] Without wishing to be limited to a single hypothesis, it is hypothesized that following ingestion of the HAV-containing transgenic tomatoes, the immune system should recognize the vital capsid proteins following the uptake of plant-derived HAV virus like particles (or similar particles applied to the rectal mucosa) by mucosal M cells which subsequently transport the virus to mucosal antigen presenting cells which process and present viral capsid peptides to mucosal helper T-cells which ultimately activate HAV-specific B and T lymphocytes in the intestinal mucosa. Using this same concept, new second-generation recombinant chimeric oral vaccines for polioviruses (also of the picomavirus family) or other viral pathogens could be developed. These would utilize HAV capsid's natural tropism for gastrointestinal epithelial cells to facilitate exposure to the mucosal immune system. These vaccines could be engineered to eliminate dangerous viral replication elements, which have, in the past, created safety issues due to spontaneous pathogenic revertants or recombination with wild type viruses present in the gut or other tissues. Such vaccines would be both safer and easier to administer than existing vaccines, as well as being relatively simple and inexpensive to produce The success of HAV particle production in transgenic plants would be the proof of principle for the production of other orally consumed viral and bacterial vaccines.

[0159] The efficient viral uptake by the gastrointestinal system could also be applied for targeting of transgenes to specific organs. The natural tropism of HAV is to the liver, probably through the expression of a viral receptor on hepatocyte cell membranes. Encapsulating the engineered HAV genome containing the desired gene in HAV nucleocapsid particles would enable targeting of genes to the liver. Similar targeting of genes to the bone marrow or the nervous system is potentially feasible using a similar strategy employing viruses displaying natural tropism for specific tissues or organs.

[0160] Another aspect of the invention pertains to the method of rectal immunization with whole virus or other immunogens to induce both local gastrointestinal) and systemic immunity against the diseases caused by viruses or other pathogens entering the body through the gastrointestinal mucosa.

[0161] Through the examples herein, mucosal vaccination has been shown to induce immunity or tolerance to HAV antigens depending upon the route of delivery (and possibly, antigen dose). Both potential outcomes of mucosal immunization could be exploited for two different treatment modalities:

[0162] 1. Generation of viral or bacterial or other structural elements in Tg plants or through other technologies for mucosal immunization to protect against infectious diseases. These genetically engineered immunogens may be used to prevent disease or to attenuate disease development. The advantage of employing pathogen structural elements as immunogens could be further developed to include multiple targets; e.g. chimeric HAV-HCV virus like particles encoding structural elements of both hepatitis A and hepatitis C viruses could be engineered in plant expression systems to serve as dual immunogens to protect against both diseases.

[0163] 2. The development of tolerance could also support various therapeutic approaches, including but not limited to:

[0164] a. Induction of tolerance to hepatitis C virus could reduce the liver inflammatory process, and reduce the risk for development of cirrhosis and hepatocellular carcinoma.

[0165] b. Induction of immune tolerance a particular virus could be exploited such that the virus could be engineered to serve as a vehicle for tissue-specific delivery of other genes in gene therapy (e.g., the use of nonpathogenic poliovirus virus like particles to target the nervous system). Immune tolerance to the viral vector would enhance its persistence in the target tissue and therefore, expression of the delivered gene.

[0166] While the invention has been described with respect to a limited number of embodiments, it will be appreciated that many variations, modifications and other applications of the invention may be made. 

1. A method for administering a viral vaccine to a subject, the viral vaccine being for a virus entering the subject through a gastrointestinal mucosa, wherein a target organ of the virus is not the intestine, the viral vaccine comprising an engineered viral particle, the engineered viral particle comprising a group of co-expressed plurality of viral proteins or portions thereof, wherein said group of co-expressed plurality of viral proteins or portions thereof is expressed in an edible plant material, the method comprising: administering the edible plant material comprising the viral vaccine to the gastrointestinal mucosa of the subject.
 2. The method of claim 1, wherein the gastrointestinal mucosa is a rectal mucosa, such that the viral vaccine is administered to the rectal mucosa of the subject.
 3. The method of claim 2, wherein the viral vaccine is in a form of a suppository.
 4. The method of claim 2, wherein the virus is HAV (Hepatitis A virus).
 5. The method of claim 4, wherein the viral vaccine contains a concentration of antigen in a range of from about 0.75 to about 7500 EL.U.
 6. The method of claim 1, wherein the subject is a lower mammal.
 7. The method of claim 1, wherein the subject is a human.
 8. The method of claim 1, wherein the viral vaccine is a commercially available vaccine originally administered by injection to the subject.
 9. A method for delivering at least one viral encapsidated gene through a gastrointestinal mucosa of a subject, the at least one viral encapsidated gene being a Hepatitis A virus (HAV) gene, the method comprising: administering the at least one viral encapsidated gene to the gastrointestinal mucosa of the subject, wherein the at least one viral encapsidated gene is contained in an engineered viral particle.
 10. The method of claim 9, wherein the gastrointestinal mucosa is a rectal mucosa, such that the viral vaccine is administered to the rectal mucosa of the subject.
 11. The method of claim 10, wherein the viral vaccine is in a form of a suppository.
 12. The method of claims 9-11, wherein the subject is a lower mammal.
 13. The method of claims 9-11, wherein the subject is a human.
 14. The method of claim 9-13, wherein the at least one viral encapsidated gene is presented in a virus-like particle, present in an edible plant material.
 15. The method of any of claims 9-14, wherein said edible material contains at least one complete viral structure as an antigen.
 16. The method of claim 15, wherein said at least one complete viral structure is a viral capsid structure.
 17. The method of claim 16, wherein said viral capsid structure is said outer capsid.
 18. A method for administering a viral vaccine for Hepatitis A virus (HAV) to a subject, wherein a target organ of HAV is the liver, the method comprising: administering the viral vaccine for HAV to a gastrointestinal mucosa of the subject, wherein the HAV is contained in an engineered viral particle, said engineered viral particle comprising a group of coexpressed plurality of viral proteins or portions thereof, wherein said engineered viral particle is expressed in an edible plant material and said edible plant material is ingested by the subject.
 19. The method of claim 18, wherein the viral vaccine contains at least one viral encapsidated gene for HAV.
 20. The method of claim 18, wherein the viral vaccine consists essentially of attenuated killed virus.
 21. The method of claim 18, wherein the viral vaccine comprises attenuated killed virus without an additional adjuvant.
 22. The method of claim 18, wherein the viral vaccine consists essentially of HAV related particles.
 23. The method of claim 18, wherein the gastrointestinal mucosa is a rectal mucosa, such that the viral vaccine is administered to the rectal mucosa of the subject.
 24. A vaccine against a disease-causing pathogen for administration to a subject, comprising: an entirety of a biologically significant structure of the disease-causing pathogen, said entirety of said biologically significant structure being expressed by an edible plant material in an engineered viral particle, wherein genes for said plurality of proteins are introduced to said edible plant material.
 25. The vaccine of claim 24, wherein said biologically significant structure is a plurality of proteins being co-expressed by said edible plant material.
 26. The vaccine of claim 25, wherein said edible plant material is transgenic for said genes, such that said genes are stably inserted into a genome of said edible plant material.
 27. The vaccine of claim 26, wherein the disease-causing pathogen is HAV (Hepatitis A virus), such that said genes are HAV genes.
 28. The vaccine of claim 24, wherein said edible plant material is administered to the subject by being eaten by the subject.
 29. The vaccine of claim 28, wherein the subject is a human being.
 30. A method for preparing the vaccine of claim 29, wherein said biologically significant structure is a plurality of proteins, the method comprising: obtaining genes corresponding to said plurality of proteins; and inserting said genes Into said edible plant material.
 31. The method of claim 30, wherein said plurality of proteins is a co-expressed group of viral proteins.
 32. The use of a vaccine against a virus, contained in an edible plant material, for oral administration to a subject, to protect the subject against the virus, wherein said edible material contains a plurality of co-expressed viral proteins or portions thereof.
 33. The use of claim 32, wherein said edible material contains at least one complete viral structure as an antigen.
 34. The use of claims 32 or 33, wherein the virus is HAV (Hepatitis A) virus.
 35. An engineered viral vaccine, comprising: a plurality of genes, including at least one viral encapsidated gene and at least one non-viral gene; and a carrier adapted to administration of the vaccine to the gastrointestinal mucosa of a subject.
 36. The vaccine of claim 35, wherein said viral sequences code for a group of co-expressed plurality of viral proteins or portions thereof.
 37. An engineered viral vaccine, comprising: at least one viral encapsidated gene; and a carrier adapted to administration of the vaccine to the gastrointestinal mucosa of a subject; wherein administration of the vaccine induces tolerance in said subject.
 38. A method for administering a viral vaccine for Hepatitis A virus (HAV) to a subject, wherein a target organ of HAV is the liver, the method comprising: administering the viral vaccine for HAV exclusively to a gastrointestinal mucosa of the subject, wherein an amount of viral particles in the viral vaccine is not greater than an equivalent amount of viral particles being: administered through intramuscular injection.
 39. The method of claim 38, wherein said gastrointestinal mucosa is a rectal mucosa.
 40. The method of claim 38, wherein the viral vaccine is administered orally.
 41. The method of any of claims 38-40, wherein said amount of viral particles is equivalent to no more than about 75 EL.U.
 42. A method for administering a viral vaccine for Hepatitis A virus (HAV) to a subject, the method comprising: administering the viral vaccine for HAV exclusively to a gastrointestinal mucosa of the subject, wherein said gastrointestinal mucosa comprises at least rectal mucosa, and wherein an amount of viral particles in the viral vaccine is in a range of from about 0.75 to about 7500 EL.U.
 43. A method for administering a viral vaccine for Hepatitis A virus (HAV) to a subject, the method comprising: administering the viral vaccine for HAV exclusively orally to the subject, wherein an amount of viral particles in the viral vaccine is in a range of from about 0.75 to about 7500 EL.U.
 44. A method for preparing a vaccine against a disease-causing pathogen for administration to a subject, the method comprising: identifying a biologically significant structure of the disease-causing pathogen, said biologically significant structure comprising a plurality of proteins wherein said plurality of proteins is a co-expressed group of pathogen proteins; obtaining genes corresponding to said plurality of proteins; and inserting said genes into an edible plant material, said edible plant material expressing said biologically significant structure in an engineered viral particle. 